RU2203944C2 - Nutrient medium for culturing and isolation of bifidobacteria - Google Patents

Abstract

FIELD: medicine, clinical microbiology. SUBSTANCE: medium comprises dry nutrient broth as source of nitrogen feeding for microorganisms, extract of fodder or bread yeast as source of vitamins of B group, trisubstituted sodium citrate, D-(+)-glucose, soda ash as solvent of medium protein components, iron sulfate, cysteine as oxidant and prooxidant, and also distilled water and microbiological agar. Components are taken in the required ratio. Invention provides improvement of growth properties of nutrient medium and can be used for culturing bifidobacteria and their quantitative determination of in diagnosis of intestine dysbacteriosis. EFFECT: improved properties of medium, simplified preparing method. 1 tbl, 6 ex

Description

 The invention relates to medicine, namely microbiology, and can be used for growing bifidobacteria and quantifying them in the diagnosis of intestinal dysbiosis.

 It is known to use casein-yeast medium and hydrolyzed milk (1, 2), Blaurock medium (modified by Goncharova) for growing bifidobacteria (3).

 The disadvantage of these environments is the complexity in their preparation in the laboratory.

 The closest solution to the problem of the same purpose to the claimed invention in terms of features is a nutrient medium for biomass accumulation, containing a source of nitrogen nutrition - an enzymatic hydrolyzate of skim milk, glucose, a source of group B vitamins - a feed concentrate of group B vitamins, corn extract, sodium tri-substituted citrate, iron sulfate 7-water, Trilon B, mannitol, agar-agar (4).

 The objective of the invention is to increase the vegetative properties of the environment and simplify the technology of its manufacture.

The specified technical result during the implementation of the invention is achieved by the fact that the proposed medium additionally contains distilled water, soda ash, as a source of nitrogen nutrition - dry broth, as a source of B vitamins - extract of feed or bread yeast, in the following ratio, g / l distilled water:
Dry nutrient broth - 20.0-30.0
D (+) glucose - 15.0-25.0
Extract of feed or bread yeast, dry - 5.0-8.0
Cysteine - 0.45-0.7
Iron sulfate 7-water - 0.13-0.18
Microbiological agar - 1.5-2.0
Sodium citrate trisubstituted - 0.8-1.2
Soda ash - 0.8-1.2
The medium is prepared as follows.

Example 1. In 1 l of distilled water, 20.0 g of nutrient broth, 15.0 g of D (+) glucose, 5.0 g of fodder yeast extract, 0.45 g of cysteine, 0.13 g of 7% aqueous iron sulfate are successively dissolved. , 0.8 g of trisubstituted sodium citrate, 1.5 g of microbiological agar, 0.8 g of soda ash. The mixture is heated, boiled for 2 minutes until the agar is completely melted, filtered through a cotton filter, poured into 9 ml sterile tubes and into 50.0 ml, 100 ml vials, sterilized for 30 minutes at 112 ^o C.

 Before use, the medium is regenerated (boiled in a water bath for 45 minutes).

 Before use, the medium is regionalized (boiled in a water bath for 10 minutes and cooled under a stream of cold water).

 To control the quality of the environment used the culture of the second generation of the lyophilized test strain of Bifidobacterium bifidum-I, obtained from GISK them. L.A. Tarasevich.

 Upon receipt of the first generation of the uterine culture, the test strain was sterilely opened and 5 ml of a sterile 0.85% sodium chloride solution was introduced into the ampoule with a pasteurized pipette.

1.0 ml of the microbial suspension was transferred to test tubes with 9 ml of the proposed and control media and titrated each medium to a dilution of 10 ^-5 to determine the viability of the B. bifidum-1 culture. The culture pipette was lowered to the bottom of the test tube with medium and the culture was gradually released, mixing the contents of the tube without aerating. During the dilution process, the microbial suspension was transferred to the next test tube using a new sterile pipette with a capacity of 1 ml.

After 48-72 h of incubation at (37 ± 1) ^o С, the growth of the culture according to the 4-point system was taken into account. Then titration of each culture was carried out before dilution of 10 ^-5 from the first tubes in the respective media. Additionally, cultures were grown (2nd generation) at the same temperature during incubation (48 ± 1) h. After incubation, the growth pattern of B. bifidum-1 culture was visually determined, and their number in dilutions with isolated colony growth was taken into account.

 The culture of bifidobacteria is characterized by the growth of colonies in a semi-liquid medium in the form of strokes from 3 mm or more in size.

 Example 2. It differs from example 1 in that the extract of bread yeast in the same (5.0) g amount is dissolved in 1 liter of water instead of the extract of feed yeast. Further according to example 1.

 Example 3. It differs from example 1.2 in that 25.0 g of nutrient broth, 20.0 D (+) glucose, 6.5 g of feed yeast extract, 0.55 g of cysteine, 0 are dissolved in 1.0 l of water. 15 g of iron sulfate 7-aqueous, 1.7 g of microbiological agar, 1.0 g of sodium trisodium citrate, 1.0 g of soda ash. Further according to example 1.

 Example 4. It differs from example 3 in that the extract of bread yeast in the same amount (6.5 g) is dissolved in 1.0 l of water instead of the extract of feed yeast. Further according to example 1.

 Example 5. It differs from examples 1 and 3 in that 30.0 g of nutrient broth, 25.0 g of D (+) glucose, 8.0 g of fodder yeast extract, 0.7 g of cysteine are dissolved in 1.0 l of water, 0.18 g of 7-aqueous iron sulfate, 2.0 g of microbiological agar, 1.2 g of trisubstituted sodium citrate, 1.2 g of soda ash. Further according to example 1.

 Example 6. It differs from example 5 in that the extract of bread yeast in the same (8.0) g amount is dissolved in 1 liter of water instead of the extract of feed yeast. Further according to example 1.

 The results of the biological control of the proposed and control environments are presented in the table.

 The table shows that the proposed environment provided a typical growth of the uterine culture of B. bifidum-1 and had better vegetative properties compared to the control environment.

 The proposed environment is economically viable, easy to prepare, does not require additional costs in the preparation. Using it in healthcare practice will help to improve the diagnosis of intestinal dysbiosis and the correct tactics of its treatment.

BIBLIOGRAPHIC DATA
1. Kalina T.E. Abstract (Rostov-on-Don), 1995

 2. Bogdanov V.M. Microbiology of milk and dairy products. - M.: Food Industry, 1969, p. 296.

 3. Epstein-Litvak R. V. Methodical recommendations. Vilshanskaya F.L. (Bacteriological diagnosis of intestinal dysbiosis). - M., 1977

 4. Ervold T. M., Perfiliev G. D., Gudkov A. V., Belova G. A. Author: certificate 789580, class C 12 N 1/20, 12.23.80.0

Claims (1)

Culture medium for the cultivation and isolation of bifidobacteria, containing a source of nitrogenous nutrition, a source of B vitamins, D (+) glucose, cysteine, trisubstituted sodium citrate, 7-hydrous iron sulfate, microbiological agar, characterized in that it additionally contains distilled water, soda calcined, as a source of nitrogen nutrition - dry nutrient broth, as a source of B vitamins - extract of feed or bread yeast in the following ratio of components, g / l distill th e water:
Dry nutrient broth - 20.0-30.0
D (+) glucose - 15.0-25.0
Dry or fodder yeast extract - 5.0-8.0
Cysteine - 0.45-0.7
Iron sulfate 7-water - 0.13-0.18
Sodium citrate trisubstituted - 0.8-1.2
Microbiological agar - 1.5-2.0
Soda ash - 0.8-1.2

Images