RU2283348C1 - Nutrient medium and preliminary identification of escherichia coli - Google Patents

Abstract

FIELD: biotechnology, in particular bacteriological detection of colon bacterium in water, foodstuff and other infected materials.

SUBSTANCE: method for identification of colon bacterium (E.coli) is based on ability of said bacterium to produce gas from lactose at 43°C in presence of boric acid. Nutrient medium contains pancreatic fish hydrolyzate as nitrous nutrition source; boric acid as selective agent; sodium hydrophosphate and potassium dihydrophosphate as buffer salts, lactose, sodium chloride and phenolic red and microbiological agar dissolved in distilled water as indicators of acid formation and gas formation indicators. Method of present invention makes it possible to reduce growth and gas-forming (caused by lactose fermentation) time of colon bacterium to 24 hours.

EFFECT: medium of improved differential properties.

1 tbl, 3 ex

Description

The invention relates to medical microbiology and can be used for bacteriological detection of Escherichia coli in food and water by its ability to grow with the formation of gas from lactose at 43 ° C in the presence of boric acid.

Currently, for this purpose, a liquid laboratory preparation of boric acid buffer medium with lactose is used (1, 2).

The disadvantage of this medium is that on this medium gas formation in the presence of boric acid (the main differentiating sign of the detection of E. coli) is observed after 48 hours of cultivation of the microorganism.

The closest solution to the problem of the same purpose to the claimed invention in terms of features is Boric Acid Broth (lactose broth with boric acid) containing proteozopeptone, lactose, potassium hydrogen phosphate, potassium dihydrogen phosphate and boric acid (3).

The boric acid buffer medium mentioned above and used in domestic microbiological laboratory practice is prepared in 1% peptone water with the addition of lactose, phosphate buffer and boric acid.

For reasons that impede the achievement of the following technical result when using a known medium, is that Boric Acid Broth provides the formation of gas from lactose after 48 hours of cultivation at a temperature of 43 ° C.

The technical result of the invention is to reduce the growth time and gas formation of Escherichia coli (as a result of lactose fermentation) up to 24 hours under the same cultivation conditions.

The specified technical result is achieved by the fact that the medium contains a pancreatic hydrolyzate of fish as a source of nitrogen nutrition, sodium hydrophosphate and potassium dihydrogen phosphate, a selective agent, boric acid, lactose, additionally contains sodium chloride, as well as microbiological and phenol red agar as indicators of gas formation and acid formation in the following ratio of components, g / l of distilled water:

Pancreatic fish hydrolyzate 9.90-10.10 Sodium chloride 4.90-5.10 Lactose 4.90-5.10 Sodium hydrogen phosphate 12.10-12.30 Potassium dihydrogen phosphate 4.10-4.30 Boric acid 3.24-3.26 Microbiological agar 1.55-1.65 Phenol red 0.075-0.085

The composition of the nutrient medium provides a high growth rate (18-20 hours) of E. coli and complete inhibition of the growth of other types of enterobacteria. The introduction of microbiological agar into the medium provides a clear capture of gas bubbles, which eliminates the need to work with glass floats and, therefore, simplify the work of the bacteriologist. The use of phenol red indicator in the medium, which is highly sensitive to changes in the pH of the medium, allows a clear visual assessment of the color change of the medium as a result of fermentation of lactose by microorganisms (E. coli).

The medium is constructed from ingredients based on dry nutrient broth having an industrial release (4).

The environment is prepared as follows.

Example 1. In a flask with 1 l of distilled water, 9.9 g of pancreatic hydrolyzate of fish, 4.9 g of sodium chloride, 4.9 g of lactose, 12.10 g of sodium hydrogen phosphate, 4.20 g of potassium dihydrogen phosphate, 3.24 g are dissolved boric acid, 1.55 g of microbiological agar, 0.075 g of phenol red.

The medium is brought to a boil, the pH should correspond to 7.0 ± 0.2 at 25 ° C, poured into sterile tubes of 9.0 ml, sterilized by autoclaving at a temperature of 110 ° C for 20 minutes. Cool to a temperature of 30-37 ° C and inoculate with test cultures. Inoculated tubes with nutrient broth are mixed and incubated at 37 ° C for 22 ± 2 hours.

Example 2. In a flask with 1 ml of distilled water, 10.0 g of pancreatic fish hydrolyzate, 5.0 g of lactose, 12.20 g of sodium hydrogen phosphate, 4.20 g of potassium dihydrogen phosphate, 3.25 g of boric acid, 1.60 g are dissolved microbiological agar, 0.080 g of phenol red.

Further according to example 1.

Example 3. In a flask with 1 liter of distilled water, 10.1 g of pancreatic hydrolyzate of fish, 5.1 g of sodium chloride, 5.1 g of lactose, 12.30 g of sodium hydrogen phosphate, 4.30 g of potassium dihydrogen phosphate, 3.26 g boric acid, 1.65 g of microbiological agar, 0.085 g of phenol red.

Further according to example 1.

Biological indicators of the proposed and known culture media for the detection and preliminary identification of Escherichia coli are presented in the table.

The table shows that the proposed environment has an advantage over the control medium prototype Boric Acid Broth and boric acid buffer medium with lactose. On these media, gas formation and acid formation (as a result of fermentation of lactose by Escherichia coli strains) is observed only after 48 hours, while on the proposed medium differentiating signs are detected after 24 hours of incubation of this type of microorganism, with complete inhibition of the growth of other types of enterobacteria. The nutrient medium is convenient and easy to use (excludes the use of glass floats), cheaper than the environment of the prototype.

Information sources

1. A.S. Labinskaya Microbiology with the technique of microbiological research. M .: Medicine, 1978, p. 385.

2. Methods of sanitary-microbiological research of environmental objects. At hand. G.I. Sidorenko, M .: Medicine, 1978, p.20-23.

3. Guidelines for the use of Himedia products in laboratory practice. 2003, p. 127-128.

4. M.M. Mejidov. Handbook of microbiological culture media. M .: Medicine, 2003, p.24-25.

Table Biological indicators of the proposed and known culture media for the detection and preliminary identification of Escherichia coli Test environment Sowing dose: the number of microbial cells in 1 ml of medium Incubation time (hour) Growth and accumulation of test cultures at a temperature of (44 ± 1) ° С Note E.coli 055 K-59 E.coli ATCC 25922 Salmonella typhi H-901 Enterobacter aerogenes No. 418 Proposed environment 10 ³ 24.0 +++ +++ - - 48.0 +++ +++ - - Boric Acid Broth Prototype 10 ³ 24.0 + + - - In the process of growth and accumulation of culture, the color of the medium does not change, because there is no indicator in the control media 48.0 ++ ++ - - Boric acid buffer with lactose 103 24.0 + + - - 48.0 ++ ++ ~ - Note: [-] - lack of growth, gas and acid formation
[+] - the presence of crop growth with turbidity
[++] - growth of crops with gas formation (without changing the color of the medium)
[+++] - crop growth with a change in the color of the medium and abundant gas formation

Claims (1)

A culture medium for the detection and preliminary identification of Escherichia coli, containing a nutrient base, lactose, sodium hydrogen phosphate, potassium dihydrogen phosphate, boric acid and water, characterized in that it additionally contains sodium chloride, phenol red and microbiological agar, and pancreatic as a nutrient base hydrolyzate of fish, water - distilled water, with the following components, g / l of distilled water: Pancreatic fish hydrolyzate 9.90-10.10 Sodium chloride 4.90-5.10 Lactose 4.90-5.10 Sodium hydrogen phosphate 12.10-12.30 Potassium dihydrogen phosphate 4.10-4.30 Boric acid 3.24-3.26 Microbiological agar 1.55-1.65 Phenol red 0.075-0.085