Nutrient medium for the isolation and cultivation of l-forms of mycobacterium tuberculosis

 

(57) Abstract:

The invention relates to veterinary Microbiology and relates to culture media to obtain the L-forms of Mycobacterium tuberculosis. The medium additionally contains unsaturated hydrocarbon with a length of chain WITH14-C17, isoniazid, and salt the buffer Hanks solution. The invention provides a reduction in terms of growth and increased biomass yield of L-forms of mycobacteria. table 2.

The invention relates to veterinary Microbiology and for the nutrient environments for experimental receive and cultivation of L-forms of Mycobacterium tuberculosis.

Known dense nutrient medium for excretion from feces of animals and cultivation of mycobacteria containing the eggs, the yolks of eggs, milk, potato broth, malachite green, potassium phosphate dvuhkamernyi, sodium citrate, magnesium sulfate, peptone, glycerol, Tetra, or Penta or hexadecan, distilled water [1].

However, the known environment is not suitable for cultivation of L-forms, as they require semi-solid nutrient medium, providing for growth of osmotichyeskoi L-forms of Mycobacterium tuberculosis, containing: the nitrogen source, sucrose, native serum of cattle or horses, sodium citrate, ammonium citrate iron, potassium phosphate, dvuhkamernyi sodium, magnesium sulfate, glycerin, agar-agar and water [2].

However, the known environment does not allow to obtain in the laboratory of experimental L-mycobacterial growth needed for experiments on the study of the characteristics caused by an infectious process. In addition, it does not provide the rapid growth of L-forms of Mycobacterium tuberculosis and sufficient accumulation of biomass, and the presence of impurities in the salt-based toxic effect on the cell.

The objective of the invention is the shortening of the growth and increase biomass yield of L-forms of mycobacteria, which is achieved by the fact that in the proposed environment salt base (prepared standard solution Hanks) is characterized by the absence of toxic ions, creates the necessary buffering and plays an essential role in metabolism. For its preparation are pre-purified reagents and deionized water.

Salt base (Hanks solution) is prepared from two main solutions “a” and “B”.

Sulfate magnesium 4,0

Water ml To 1000

Sterilized, avtoclaviruut 20 min 1.5 ATM.

The main solution “B”, g:

Sodium bicarbonate 7,0

Disubstituted phosphate sodium 1,2

One-deputizing potassium phosphate 1,2

Glucose 20

Water ml To 1000

Solution “B” is sterilized by filtration through a plate EC2in the Seitz filter.

To 900 ml of distilled water add 50 ml of solution “A”, autoclave and aseptically add 50 ml of solution “B”. Set pH 7,2 - 7,3 by adding sterilized by filtration of a 1.5% solution of sodium bicarbonate.

As a growth stimulator nutrient medium contains one of saturated hydrocarbons with chain length WITH14-C17. Saturated hydrocarbons with a chain length of C14-C17in a dose of 0.1-0.2 ml per tube have a growth promoting effect, actively affect metabolic processes in microbial cell, stimulates the synthesis of carbohydrates are the energy source and the plastic material, the lower dose does not provide the growth-promoting effect and increased doses leads to a decrease in the rate of growth, so this amount (0.1-0.2 ml) is what keeps isoniazid, which affects protein synthesis of mycobacterial cells, resulting in the violation of the integrity of the cell wall of bacteria and the formation of L-forms.

Pathogenic mycobacteria are biochemically less active, as evidenced by their slow reproduction and the need for a complex of nutrients. They contain less enzymes and growth substances, therefore, are more sensitive to antibiotics.

You must have in the native environment of the blood serum of cattle or horses. It has a buffer properties, and also has a stimulating effect on the reproduction and growth of L-forms, as with the serum in the medium receives proteins and a number of strongly related vitamins.

Example 1

It was tested the influence of different doses of isoniazid on cultures of M. bovis units 8 and M. avium pieces 9. Crops were sown on the tested nutrient semisolid medium. The results of the study are presented in table 1.

At carrying out of smear microscopy, we observed that in test tubes containing 0.5 μg/ml of isoniazid both cultures were represented typical cells (97-100%).

At a concentration of 1 MK is vacuolation cells (37-43%) granular rods and spherical bodies (10-20%). M. avium pieces 9 in this case was also presented a typical cell (93%).

When the concentration is increased in 2 times typical cells of M. bovis units 8 was absent, modified forms was observed in acceptably cells (55%), in test tubes with M. avium pieces 9 there was a great number of typical cells (68%), but was observed and modified forms, mainly granular coli (23%).

With the increasing content of up to 5 µg/ml of the growth of M. bovis 8 was absent, and M. avium 9 grown in mixed culture: typical 60% and changed to 40%. And only by increasing the concentration of isoniazid 10 mg/ml in the culture of M. avium 9 - was not observed typical cells.

Thus, we have experimentally found that isoniazid has on pathogenic mycobacteria L-transforming action in a dose of 1 µg/ml of medium. Mycobacterium avium species and atypical mycobacteria synthesize more growth substances and vitamins, therefore, chemotherapeutic substance acts on them in higher concentrations (10 µg/ml). Therefore isoniazid in the proposed environment is used in a dose of from 1 to 10 µg/ml, which is effective to achieve its goals.

Cooking environment. A portion of the agar-agar (0.3-0.4 grams) was diluted (fueling the -2,2 ml), isoniazid (1-10 μg/ml) to bring the volume to 100 ml with Hanks solution and autoclave at 0.5 ATM. 30 minutes medium is poured into 9 ml in a test tube and incubated at 37°C for 2-3 days to check for sterility. Store the environment in a well-sealed tubes at 0 - (+5)°C for 3-4 weeks. Before planting add native serum of cattle or horses (10-20 ml).

The proposed nutrient medium is different from the known fact that additionally contains unsaturated hydrocarbon long chain WITH14-C17, isoniazid, and salt the buffer solution Hanks in the following ratio of components:

Agar-agar, g 0,3-0,4

Native serum

cattle

or a horse, 10-20 ml

Sucrose 200%, ml 9-12

Unsaturated hydrocarbon

with a length of chain WITH14-C17, ml 1,1-2,2

Isoniazid, ug/ml 1-10

Salt base - buffer

the Hanks solution, ml To 100

Example 2.

To prepare 100 ml of culture medium take the following ingredients:

Agar-agar, g 0,3

Sucrose 200%, ml 9

Pre-Christ.

cattle

or a horse, ml 10

Isoniazid, ug/ml 1

Salt base - buffer

the Hanks solution, ml To 100

A portion of the agar-agar is dissolved in saline basis, then add sucrose 200%, hydrocarbon, isoniazid and autoclave at 0.5 ATM 30 minutes After cooling, add native serum of cattle or horses. The nutrient medium is poured into 9 ml in a test tube, incubated at 37°C for 2-3 days to check for sterility. Store the environment in a well-sealed tubes at 0 - (+5)°C for 3-4 weeks.

Example 3.

Prepare the environment in accordance with example 2, but the components are taken in the following amounts:

Agar-agar, g 0,4

Sucrose 200%, 12 ml

Unsaturated hydrocarbon

with a length of chain WITH14-C17, ml 2,2

Native serum

cattle

or a horse, ml 20

Isoniazid, ug/ml 10

Salt base - buffer

the Hanks solution, ml To 100

The results of the test media prepared in examples 1 and 2 for the cultivation of L-forms of various kinds mycobateria the use of the proposed nutrient medium can accelerate the growth of L-forms in 2-3 times, to increase the intensity of accumulation of biomass in 2 times.

Thus, the use of the new environment and achieve the objectives of the invention, which allows to make a conclusion on the conformity of the proposed solutions to the criterion of “positive effect”.

The set of distinctive features meets the criterion of “novelty” and “inventive step”.

Sources of information

1. A. S. No. 2059728, C 12 Q 1/04. Nutrient medium for excretion from feces of animals and cultivation of Mycobacterium tuberculosis. Authors: Hodon L. M., Thaler L. A. and others

2. Dorokhova I. R., Kochemasov H. N., Digno M M Selection of L-forms of Mycobacterium tuberculosis from pathological material / guidelines, M., 1984.

Nutrient medium for the isolation and cultivation of L-forms of Mycobacterium tuberculosis containing sucrose, native serum of cattle or horses, agar-agar, salt base, characterized in that it further contains an unsaturated hydrocarbon with a length of chain WITH14-C17, isoniazid, and as salt basics - buffered Hanks solution in the following ratio of components:

Agar-agar, g 0,3-0,4

Native savor the unsaturated hydrocarbon

with a chain length of C14-C17, ml 1,1-2,2

Isoniazid, ug/ml 1-10

Salt base - buffer solution

Hanks, ml To 100



 

Same patents:

The invention relates to biotechnology, relates to a method for the study of microbes, mainly in food products for contamination of pathogens and other microbes, detection and/or identification of bacteria and other microbes

The invention relates to biotechnology
The invention relates to Microbiology, and in particular relates to a method of obtaining nutrient media, providing optimal conditions for vital activity of V. cholerae can be used in Microbiology and biotechnology
The invention relates to biotechnology and sanitary Microbiology, in particular, is concerned with the obtaining of a nutrient medium for the accumulation of biomass vaccine strain of the plague microbe

The invention relates to biotechnology and can be used in meat industry.

The invention relates to biotechnology and can be used for microbial protein

The invention relates to biotechnology, in particular relates to a method of obtaining nutrient media, providing optimal conditions for vital activity of Brucella bacteria, and can be used in medical Microbiology

The invention relates to medicine, namely Microbiology, and can be used in medical practice for the detection of Salmonella in food products, the objects of the external environment, as well as in faeces from patients with salmonellosis adults and children

The invention relates to biotechnology, namely, clinical Microbiology, and can be used for bacteriological diagnosis of diseases caused by bacteria of the genus Yersinia

The invention relates to Microbiology and ecology, in particular to methods of selection odnopestichnyj options anthrax in the S-form
The invention relates to Microbiology, and in particular relates to a method of obtaining nutrient media, providing optimal conditions for vital activity of V. cholerae can be used in Microbiology and biotechnology
The invention relates to biotechnology and sanitary Microbiology, in particular, is concerned with the obtaining of a nutrient medium for the accumulation of biomass vaccine strain of the plague microbe

The invention relates to applied Microbiology and can be used for cleaning the surface of the water from spilled oil and petroleum products

The invention relates to the field of biotechnology, and more particularly to a strain of Shigella sonnei No. 262 and can be used to produce vaccine and diagnostic products

The invention relates to biotechnology and can be used in meat industry.

The invention relates to biotechnology and can be used for microbial protein

The invention relates to agriculture, in particular animal feed production, and can be used in the ensiling of green mass feed

The invention relates to biotechnology, in particular relates to a method of obtaining nutrient media, providing optimal conditions for vital activity of Brucella bacteria, and can be used in medical Microbiology

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

Up!