Nutrient medium for growing legionella

FIELD: biotechnology.

SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.

EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

 

The invention relates to biotechnology, in particular to obtain nutrient media for cultivation of Legionella.

Known nutrient medium for cultivation of Legionella, which is based on meat water to 1000,0 ml; peptone is an enzymatic - 10.0 g; sodium chloride 5.0 g; blood, 10 g; agar-agar - 15,0-20,0 g; pH 7,3±0,2. Wednesday autoclave for 30 min at 121°C (Mosk; Diotalevi; Meachanic. Nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb.-2008.-P.64-65.-C.-269).

The disadvantage of this environment is the lack of efficiency.

Closest to the proposed nutrient medium is a medium containing, g/l: enzymatic hydrolysate easy pig - 260,0; potassium phosphate 1-substituted - 0,9; potassium phosphate 2-substituted 3-water - 2,2; activated charcoal - 2,0; L-cysteine hydrochloride - 0,4; embryonic growth of microorganisms is 2.5; the microbiological agar - 8,5; distilled water to 1 l (RF Patent No. 2412240. Publ. 20.02.2011, bull. No. 5).

The disadvantage of this environment is multi-stage and the difficulty of obtaining embryonic growth of microorganisms.

The aim of the present invention is the construction quality and easy to prepare nutrient environment of animal origin, which provides growth properties Legionella in 36 hours

<> This objective is achieved in that the culture medium as a nutrient basis contains the enzymatic hydrolysate of light pigs; 0.01 M potassium phosphate buffer (potassium phosphate buffer: potassium phosphate 1-substituted; potassium phosphate 2-substituted 3-water), as well as the coal is active, L-cysteine hydrochloride; distilled water, the microbiological agar with the addition on Wednesday as growth stimulator enzymatic hydrolysate yolk of chicken eggs in the following ratio of ingredients, g/l:

Enzymatic hydrolysate of the lung of a pig
with the content of amino nitrogen 0,10-0,14%240,0-280,0
Enzymatic hydrolysate of yolk of egg3,0-7,0
Potassium phosphate 1-substituted0,7-1,1
Potassium phosphate 2-substituted
3-water2,0-2,4
Coal active1,0-3,0
L-cysteine hydrochloride0,2-0,6
Agar Microbiology6,5-10,5
Distilled waterto 1 l

Light pigs TU-8-5344-113607. The biological value of the lung of a pig-related products category II is available in an average of 13.6% protein, minerals (salts of sodium, potassium, calcium, phosphorus) and high iron content; their total number reaches 1%, 0.5 to 1.4 per cent ash. Light also contains vitamins, minerals, extractive substances. Of the proteins in the lung of a pig in enzymatic hydrolysis causes the following amino acids: lysine, methionine, tryptophan, cystine, threonine, serine, phenylalanine, Proline, alanine, glycine, valine, leucine, tyrosine, histidine (Ivithin. Feeds and feed additives. Moscow. Rosagropromizdat. 1989. C.-104-108).

The biological value of egg yolk of chicken eggs is an average of 50% water, protein is 16.2%, amino acids, macro-elements in mg per 100 g: potassium - 129; calcium - 129; magnesium - 15; sulfur - 170; chlorine - 146,8; phosphorus - 542; sodium - 51, trace elements in µg per 100 g: iron - 6700; iodine - 23; cobalt - 23; manganese - 37; copper - 139; molybdenum - 11,8; chromium - 7,8; zinc - 3105. The content of vitamins mg per kg: vitamin a - 0,35; carotene - 0,06; vitamin B1- 1,6; vitamin D - 4,70; vitamin E - 2,0; Pantothenic acid - 1,3; vitamin b5- 3,0;6to 0.14; Niacin - 019; Riboflavin - 0,44; folacin - 7,0; choline - 251,7. The conversion of the essential amino acids in g/kg is 6558: valine - 937; isoleucine - 907; leucine - 1381; lysine - 1156; methionine - 415; threonine - 830; tryptophan - 236 and phenylalanine - 696. The conversion factor of essential amino acids is 9331, including: alanine - 854; arginine - 1156; aspartic acid - 1339; histidine - 383; glycine -514; glutamic acid - 2051; Proline - 695; serine - 1365; tyrosine - 699; cystine - 275. Trace elements mg / kg: zinc - 14,0; manganese - 0,8; copper - 0,8; cobalt - 0,07. Organic substances: protein - 93% (protein); fat - 94% (Mfreeman, Imekura. The chemical composition of food. Moscow. "Food industry". 1979. 247).

L-cysteine hydrochloride - amino acid (Panreac 15 W/ Batch Lot 0000054464), which accelerates the growth of Legionella.

Hydrochloric acid GOST - 3118-77 (analytical grade, reagent-grade) is used to measure the pH. Mass fraction of basic substance, % 35-38.

The microbiological agar - agar bacteriological (European type). Manufacturer: "Pronadisa" Spain. Party LF 15110173. Date of manufacture: April 2012

Coal active charcoal powder, brand OS-A. Fine powder, black. Without foreign inclusions, corresponds to GOST 4453-74. Absorbs in himself the waste products of metabolism of Legionella.

Legionella are optional nutrici the internal parasites so grow in the yolk bag of chicken embryos in culture of animal cells and human lung fibroblasts). (Medical Microbiology, Virology and immunology: a Textbook for medical students /edited by Bev. - 2nd ed., Corr. and extra - M.:OOO "Medical news Agency", 2008. - C.-400).

The inclusion in the composition of the medium potassium phosphate buffer 0.01 M (composition potassium phosphate buffer: potassium phosphate substituted 1; potassium phosphate 2-substituted 3-water) justified the extensive use of phosphates in the preparation of nutrient media, because it is the only inorganic compounds having a buffer action in the physiologically important range of pH, they have low toxicity (Methodical recommendations for the production and use of culture media and solutions for microbiological purposes, cultivation of cells and viruses. -M., 1989). (Mosk; Diotalevi; Meachanic. Nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb.-2008. - P.37-38).

In the preparation of the nutrient medium is allowed to use only distilled water without the use of solutions containing sodium ions, because of their destructive actions.

Unlike the prototype, the combined use of these components provides obtaining pure cultures after 36 hours

Preparation, is the enzymatic hydrolysate of pig lung.

Easy pigs in a quantity of 1 kg cut into pieces of size 2x2 cm poured into an enamel saucepan, pour tap water in the amount of 1.5 liters, boiled for 10 min, cooled to 46°C. the Broth is poured into a 3 l glass bottle. The cooled pieces of light passed through a meat grinder, then placed in the container with the broth. The enzyme Pancreatin in the amount of 9.5 g/l (or the pancreas of cattle, missed twice through a meat grinder in the amount of 150 g/kg) is placed in the container with meat and light broth. Bring pH to 8.2±0,1 using potassium hydroxide to phenolphthalein. As preservative added chloroform in the amount of 1% by volume of the liquid. The container is closed with a cotton-gauze tube, mix thoroughly and placed in a heat chamber at a temperature of 37±1°C. the Contents of the container during the day mix, every 20±1 min for 5 min, and in the next day after 1.5±0.2 h for 5 minutes Dynamics of the process of hydrolysis is controlled by determining the amino nitrogen. About the end of the hydrolysis judged by the achievement of the amine nitrogen of 0.5%and-0.6% (after 5 days). After this the heating and stirring is stopped, the hydrolysate defend. Settled the upper layer is decanted from the hydrolysate by means of a rubber hose and filtered through a cloth filter. The leachate lead in a glass container with a capacity of 3 l, to conserve add hloroform in the amount of 1% by volume of the contents of a container, closed with a rubber stopper and transported in a cold chamber, where it is stored at a temperature of from +2 to +8°C.

Preparation of enzymatic hydrolysate of yolk of egg

The yolks of chicken eggs in the quantity of 30 pieces, that is 550 ml, poured into a 3-liter glass container, pour warmed up to 42+1°C drinking water to 1.5 l, alkalinized with 20% solution of potassium hydroxide at a rate of 13 ml) to a pH of 8.2 to phenolphthalein, then add commercial enzymes: trypsin in the number of 31.0 g and Pancreatin - 10,0 g as preservative added chloroform in the amount of 1% by volume of the liquid. The container is closed with a cotton-gauze tube, mix thoroughly and placed in a heat chamber at a temperature of 42°C. the Contents of the container during the first day is stirred in 20±1 min 5 min later, after 1.5-2.0 hours and 5 minutes Daily is the measurement of pH and increase of the amine nitrogen. Two days due to a sharp drop of pH to 5% add 30 ml of 20% potassium hydroxide to a pH of 8.2 to phenolphthalein. The dynamics of the process of hydrolysis is controlled by determining the amino nitrogen. About the end of the hydrolysis judged by the achievement of the amine nitrogen 0,7%-0,8%. After this the heating and stirring is stopped, the hydrolysate assert within 2 days. Settled the upper layer is decanted from the hydrolysate by means of a rubber hose and filter is via a plain filter. The leachate lead in a glass container with a capacity of 3 l, to conserve add chloroform in the amount of 1% by volume of the contents of a container, cover with a rubber stopper and transported in a cold chamber, where it is stored at a temperature of from +2 to +8°C.

Nutrient medium for the enzymatic basis of light pigs for cultivation of Legionella is prepared in the following way. Take the enzymatic hydrolysate of lightweight pigs, diluted with distilled water to the testimony of amino nitrogen 0,12% - 260,0 ml; enzymatic hydrolysate of yolk eggs - 5.0 g/l; potassium phosphate 1-substituted - 0.9 g/l; potassium phosphate 2-substituted 3-water and 2.2 g/l; the active coal - 2.0 g/l; agar microbiological - 8.5 g/l; distilled water to 1 L. the Mixture is brought to a boil and boil until complete melting of agar for 2 min, then add 0.4 g of L-cysteine hydrochloride dissolved in 10 ml of distilled water, pH adjusted with hydrochloric acid (1:1) to 6.9±0.5 V quantity of 2.2 ml of Prepared nutrient medium is poured into a graduated vials of 200±10.0 ml, which is hermetically sealed cotton-gauze tubes and paper Craft. Sterilized at 1 ATM for 20 minutes After melting in a boiling water bath and cooled to 56°C, the agar is poured into sterile Petri dishes. Dried for 30 min and used for planting

The efficiency obtained nutrient medium was evaluated in accordance with the methodological instructions "Control diagnostic culture media for biological indicators for plague, cholera, anthrax, tularemia, brucellosis, legionellosis" (Moscow, 2006), using as the test cultures Legionella Legionella pneumophila was ATSS 33155 Bloomington, Legionella pneumophila ATCC 33152 Philadelphia.

The subjects of culture was grown on plates of solid agar pH 6.8 at a temperature of 37°C 24 h Of overnight cultures were prepared 1 billion suspension M.K. 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 of 100 M.K. data From dilutions of suspensions of crops were sown in 0.1 ml of 3 Petri dishes spilled dried agar. Crops were incubated at 37°C for 5 days with daily viewing. Through 24-120 h took into account the results by calculations of grown colonies.

Example 1. The test strains were grown on nutrient medium containing, g/l: enzymatic hydrolysate of lightweight pigs, diluted with distilled water to the testimony of amino nitrogen 0,100% - 240,0 ml; enzymatic hydrolysate of yolk eggs - 3.0 ml; potassium phosphate 1-substituted - 0,7; potassium phosphate 2-substituted 3-water - 2,0; angle active ,0; L-cysteine hydrochloride and 0.2; the microbiological agar - 6,5; distilled water to 1 L. this ratio of ingredients cumulative effect was less than 50%, after 36 hours of grown colonies from sowing the test strains in the amount of 2 (PLANO-convex, bluish-grayish diameter of 1.0-1.1 mm), whereas the control environment, the SAL growth was observed only after 72 h on agar of Hottinger growth was not observed.

Example 2. The test strains were grown on nutrient medium containing, g/l: enzymatic hydrolysate of lightweight pigs, diluted with distilled water to the testimony of amino nitrogen 0,12% - 260,0 ml; enzymatic hydrolysate of yolk eggs - 5.0 ml; potassium phosphate 1-substituted - 0,9; potassium phosphate 2-substituted 3-water - 2,2; angle active - 2,0; L-cysteine hydrochloride - 0,4; microbiological agar - 8,5; distilled water to 1 L. this ratio of ingredients cumulative effect exceeds 50%, the grown colonies from sowing test strains in the number 7 (PLANO-convex, bluish-grayish colonies with a diameter of 1.2-1.5 mm) through 36 h, whereas the control environment, the SAL growth was observed only after 72 h on agar of Hottinger growth was not observed.

Example 3. The test strains were grown on nutrient medium containing, g/l: enzymatic hydrolysate of lightweight pigs, diluted with distilled water to show the Oia amine nitrogen of 0.14%-280,0 ml; enzymatic hydrolysate of yolk of eggs of 7.0 ml; potassium phosphate 1-substituted - 1,1; potassium phosphate 2-substituted 3-water - 2,4; the active coal - 3,0; L-cysteine hydrochloride - 0,6; microbiological agar - 10,5; distilled water to 1 L. this ratio of ingredients cumulative effect was observed less than 50%, the grown colonies from sowing the test strains in the amount of 3 (PLANO-convex, bluish-grayish diameter of 1.0-1.1 mm) through 36 h, whereas the control environment, the SAL growth was observed only through 72 hours on agar of Hottinger growth was not observed.

The obtained results allowed to determine which option is best nutrient medium for the enzymatic basis of light pigs (example 2).

The proposed environment quality, easy to prepare, based on the enzymatic hydrolysate of light pigs, and as a stimulator of growth of Legionella - enzymatic hydrolysate yolk of chicken eggs. Profitability is 29%.

Nutrient medium for cultivation of Legionella-containing, g/l:

Enzymatic hydrolysate of the lung of a pig
with the content of amino nitrogen 0,10-0,14%240,0-280,0
Enzymatic g is kalisat yolk of chicken eggs 3,0-7,0
Potassium phosphate 1-substituted0,7-1,1
Potassium phosphate 2-substituted
3-water2,0-2,4
Coal active1,0-3,0
L-cysteine hydrochloride0,2-0,6
Agar Microbiology6,5-10,5
Distilled waterto 1 l



 

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