Transport nutrient medium (liquid) for cultivation of brucellous microbe

FIELD: medicine.

SUBSTANCE: nutrient medium contains yeast water, beef hydrolyzate, sodium chloride, glucose, glycerin, sodium citrate, sodium metabisulphite and distilled water.

EFFECT: invention allows providing optimum conditions for brucellous microbe growth, replication in a transport nutrient medium at any distances.

3 ex

 

The invention relates to medical Microbiology, in particular the production of culture media, providing optimal conditions for life, transportation and cultivation of Brucella organism.

Known nutrient medium, which is based on the agar Martin, of the following composition, g/l: peptone Martin 0,5; sodium chloride 3,0; 20% sodium hydroxide 3,0; agar microbiological 2,0; meat water of 0.5; pH 7,2±0,1 (Circassian F.K., Epiphany LB, Belsky N.A. Microbiology. - M.: Medicine, 1987).

The disadvantage of this environment is its lack of productivity.

Closest to the proposed nutrient medium is a liquid transport medium for the collection, cultivation and transportation of blood samples of patients suspected to be infected with brucellosis, containing, g/l: hydrolyzed beef 170,0; sodium chloride 5,0; glycerin 20,0; glucose 10.0 g; sodium citrate 5,0; SAG-1 0,6; 20%sodium hydroxide 0,001-0,0003, distilled water to 1 l (RF patent No. 2247775. Publ. 10.03.2005, bull. No. 7).

The disadvantage of this environment is the inability of acquisition growth stimulator Brucella SAG-1 due to the closure of the Institute of Bioorganic chemistry, USSR Academy of Sciences (Ukraine).

The aim of the invention is to obtain nutrient transport (liquid) for the cultivation of Brucella organism.

the left goal is achieved by what nutritional basis of the proposed nutrient transport (liquid) for the cultivation of Brucella organism is hydrolyzed beef, the environment also includes yeast water, sodium chloride, glucose, glycerol, sodium citrate, distilled water is added to the environment stimulating additives - sodium metabisulfite, in the following ratio of ingredients, g/l:

Yeast water18,0-22,0
Hydrolyzed beef70,0-110,0
Sodium chloride3,0-7,0
Glucose8,0-12,0
Glycerin18,0-22,0
Sodium citrate3,0-7,0
Metabisulphite sodium0,1-0,5
Distilled waterto 1 l

To obtain yeast water is used yeast baking pressed GOST 171-81 (manufacturer LLC Drageevic", Karachay-Cherkessia, Adyge-Halske R-n, Erkin-Saharsky sugar factory). Raw material for production of bread yeast are the two who is molasses beet (sugar) - waste sugar beet production (beet molasses), which comprises heat-resistant, biologically-active substances, which become yeast. In addition, they contain vitamins b3B8and N-Biotin are essential vitamins for growth. Also in yeast contains trace elements: boron, fluorine, aluminum, phosphorus, manganese, iron, copper, molybdenum, and amino acids: glycine, alanine, valine, leucine, isoleucine, series, aspartic, glutamic, tyrosine, Proline - other amino acid found in trace amounts (Bolotov N.A., Faradzev DU Manufacture of bakery yeast. M: Profession, 2002. - 167 C.; Semikhatova NM Production of yeast. M: Food industry, 1967. - 154 C.).

Used metabisulfite sodium, according to the authors Zo Bell S.E. and K.F. Meyer (1932) (Lankford et al., 1952; Lankford et al.,1956; Rode et al., 1951; Guzowski A.G. (1982)), fully meets the needs of brucellosis microbe in the presence of sulfur drug.

The proposed environment provides optimal conditions for growth and reproduction brucellosis microbe. Using yeast water, as well as the introduction of the environment stimulating additives of sodium metabisulfite completely replace SAG-1, amplify the growth properties of the nutrient base - hydrolyzed beef - and are hallmarks discov is Oh nutrient medium.

The method of preparation of the nutrient medium of transport for the diagnosis of brucellosis microbe is as follows: bread yeast in the amount of 1 kg was dissolved in 2 l of distilled water, heated to dissolve the yeast, boiled for 5 min, then filtered through the canvas Belting. Ready yeast water in the amount of 20.0 ml add at 90.0 ml of hydrolysate beef, diluted with distilled water to a content of amino nitrogen of 100 mg % and boiled to remove chloroform, and then add sodium chloride 5.0 g; sodium citrate 5.0 g, boil to dissolve all components, set pH 7,3±0,1 sodium hydroxide 20%. The precipitation is filtered through a fabric filter with a filter paper, add glucose 10.0 g; glycerol 20,0 ml; sodium metabisulfite 0,3, Wednesday poured into sterile penicillin vials V 15 ml of 5.0±0.1 ml, sealed with a sterile rubber stoppers, cover with aluminum caps, rolls, sterilized at 1 ATM 15 minutes

The effectiveness of the proposed nutrient medium was evaluated in accordance with the methodological instructions "Control diagnostic culture media for biological indicators for the plague, cholera, anthrax, tularemia, brucellosis, legionellosis", MU 3.3.2.2124-06, Moscow, 2007, using as the test cultures the Ref is entih virulent strains of Brucella B. melitensis 16 M; B. abortus 544. The subjects of culture was grown on plates of solid agar pH 7.3±0,1 at a temperature of 37°C 48-120 hours From a two-day cultures were prepared 1 billion suspension M.L. test strains equal to 10 units of optical turbidity standard of CCA gisk named after. Lautareasca, then serial tenfold dilutions in physiological solution of 4.5 ml conveyed to the content in 1 ml 1000; 500; 100 M.K. From each dilution of the suspension cultures were sown disposable syringes 0.1 ml 3 bottles. Crops were incubated at a temperature of 37±1°C for 5 days. Through 48-72-120 h took into account the number of grown colonies.

Example 1. Reference strains were grown on nutrient medium containing, g/l: yeast water 18,0; hydrolyzed beef 70,0; sodium chloride 3,0; glucose 8,0; glycerin 18,0; sodium citrate 3,0; sodium metabisulfite 0,1; distilled water to 1 L.

With this ratio of ingredients, the number of colonies grown on solid agar plates after 48 h, for brucellosis microbe B. melitensis 16 M and B. abortus 544 when planting a dose of 50 and 10, M.K. amounted to 290; 248; 201 and 135; 129; 127 colonies in S shape with a diameter of 1.3 mm, while sowing the dose of 100 M.K. on plates of agar was obtained a solid growth of bacteria.

Example 2. Reference strains were grown on nutrient medium containing, g/l: yeast water 20,0; hydrolyzed beef 90,0; sodium chloride 5,0; glucose 10, 0mm; glicerin,0; sodium citrate 5,0; metabisulphite sodium 0,3; distilled water to 1 L.

With this ratio of ingredients, the number of colonies grown on solid agar plates after 48 h, for brucellosis microbe B. melitensis 16 M and B. abortus 544 when planting a dose of 50 and 10, M.K., respectively 378; 350; 310 and 190; 159; 130 colonies in S shape with a diameter of 1.5 mm, while sowing the dose of 100 M.K. on plates of agar was obtained a solid growth of bacteria.

Example 3. Reference strains were grown on nutrient medium containing, g/l: yeast water 22,0; hydrolyzed beef 110,0; sodium chloride 7,0; glucose 12,0; glycerin 22, 0mm; sodium citrate 7,0; metabisulphite sodium 0,5; distilled water to 1 L.

With this ratio of ingredients, the number of colonies grown on solid agar plates after 48 h, for brucellosis microbe B. melitensis 16 M and B. abortus 544 when planting a dose of 50 and 10, M.K. amounted to 260; 227; 210 and 112; 100; 99 colonies in S shape with a diameter of 1.3 mm, while sowing the dose of 100 M.K. on plates of agar was obtained a solid growth of bacteria.

Thus, a variant of the obtained culture medium (example 2) is the optimal number of carefully selected ingredients, they collectively provide for the growth of cultures of B. melitensis 16 M and B. abortus 544 and allow you to obtain a final concentration of bacteria after 48 h, significantly exceeding the sowing dose.

Nutrient medium No. 2 in the share of the ve 5 ml placed in penicillin vials V 15 ml, sealed with rubber plugs, cover with aluminum caps, rolls, sterilized at 1 ATM for 15 min and used for the introduction of blood at the bedside of patients suspected to be infected with brucellosis, and transported to any distance.

Nutrient medium transport (liquid) for the cultivation of Brucella microbe, comprising, g/l:

Yeast water18,0-22,0
Hydrolyzed beef70,0-110,0
Sodium chloride3,0-7,0
Glucose8,0-12,0
Glycerin18,0-22,0
Sodium citrate3,0-7,0
Metabisulphite sodium0,1-0,5
Distilled waterRest



 

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