Nutrient medium for legionella cultivation

FIELD: medicine.

SUBSTANCE: invention can be used in producing nutrient mediums which create the optimum conditions of legionella activity. The nutrient medium contains a enzymatic hydrolyzate of a pig's lung, a enzymatic hydrolyzate of peanut, 1-substituted potassium phosphate, 2-substituted 3-aqueous potassium phosphate, activated carbon, L-cysteine hydrochloride, microbiological agar and distilled water.

EFFECT: invention allows reducing time for legionella detection.

3 ex

 

The invention relates to biotechnology, namely, 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 g; sodium chloride 5.0 g; blood 10 g; agar-agar 15,0-20,0 g, pH 7.3 ą 0.2. Wednesday autoclave 30 min at 121°C (Mosk; Diotalevi; Meachanic. Nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb. - 2008. - P.64-65; S).

The disadvantage of this environment is the lack of efficiency.

Closest to the proposed nutrient medium is a medium containing g/l: yeast extract - 10.0 g; activated carbon - 2.0 g; ACES buffer (amino-2-oxyethyl-aminoethane sulfonic acid) - 10.0 g; a-Ketoglutarate - 1.0 g; L-cysteine - 0.4 g; ferric pyrophosphate, 0.25 g; agar-agar 15.0 g; distilled water to 1 l; pH of 6.9 ą 0.2. (Mosk, Diotalevi, Meachanic. Nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb. - 2008. - S-270).

The disadvantage of this environment is to expensive not produced in the Russian Federation drug ferric pyrophosphate soluble.

The aim of the present invention is the construction quality of the nutrient medium of animal origin, which provides growth properties Legionella.

This goal is achieved by which the nutrient medium as a nutrient basis contains the enzymatic hydrolysate of the lung of a pig; 0.01 M califofnia buffer (composition califofnia buffer: potassium phosphate 1 - substituted; potassium phosphate 2-substituted 3-water); and activated carbon; L-cysteine hydrochloride; distilled water; microbiological agar with the addition of Wednesday stimulator enzymatic hydrolysate of peanuts in the following ratio of ingredients, g/l:

Enzymatic hydrolysate of the lung of a pig260,0
Potassium phosphate 1-substituted0,9
Potassium phosphate 2-substituted 3-water2,2
Activated carbon2,0
L-cysteine hydrochloride0,35
Enzymatic hydrolysate of peanuts10,0
Agar Microbiology10,0
Distilled water up to1 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, mineral substances is easy, consisting of 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, cysteine, threonine, serine, phenylalanine, Proline, alanine, glycine, valine, leucine, tyrosine, histidine. (Ivithin. Feeds and feed additives. Moscow: Rosagropromizdat. C.-104-108).

The inclusion of the environment in 0.01 M califofnia buffer (composition califofnia buffer: potassium phosphate acid substituted 1; potassium phosphate 2-substituted 3-water); grounded 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 pH range, they have low toxicity. In addition, HPO4is a component of nucleic acids, phospholipids, nucleotides (Methodical recommendations for the production and use of culture media and solutions for microbiological purposes, cultivation of cells and viruses - M., 1989).

Enzymatic hydrolysate of peanuts is a stimulant nutrient medium due to the presence of L-cysteine. Determination of L-cysteine and including other amino acids - lysine is alankila, arginine, glutamine, asparagine, phenylalanine, valine, ilatina contained in peanuts, carried out using the method of thin-layer hromatography on the plates of Silufol. (Aphorisms fundamentals of analytical chemistry. Physico-chemical (instrumental) methods of analysis. Ed. "Chemistry". 1970. - 472 S.)

In the preparation of nutrient media use only distilled water, without the use of solutions containing sodium ions, because of their destructive actions on Legionella.

The combined use of these components provides obtaining pure cultures after 48 hours

Legionella are facultative intracellular parasites, therefore, 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. - S.400).

Unlike the prototype of the proposed environment economical, profitable and provides optimal conditions for the proliferation of Legionella in 48 hours

Preparation of enzymatic hydrolysate of the lung of a pig

Easy pigs in a quantity of 1 kg cut into pieces 2×2 cm poured into an enamel saucepan, pour tap water in the amount of 1.5 liters, cooking is within 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 based 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 first day is stirred in 20±1 min to 5 min, in the next 1.5-2.0 hours and 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 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 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.

The enzymatic hydrolysate peanuts is prepared as follows:

seeds of peanuts in the amount of 0.5 kg is thoroughly washed in running potable water (pre-placing them in cheesecloth). The washed seeds peanuts crushed in a grinder, put in a glass container and add 1.5 l of drinking water in the ratio (1:3), warmed to a temperature of 45±1°C. Adjusted the pH to 8.2±0,3 using potassium hydroxide in the amount of 3.0±0.1 g/l to phenolphthalein. Then download 0.27 kg twice filtered through a meat grinder pancreas of cattle. To the volume of the contents of a container add 1% chloroform. The container is closed with a cotton-gauze tube with parchment, thoroughly mixed and placed in a heat chamber at a temperature of 45±1°C. the Contents of the container during the first day, mix 15±1 min for 5 min, and later 2.0 hours and 5 minutes Dynamics of the enzymatic process is controlled by the increase of amine nitrogen. About the end of the hydrolysis judged by the achievement of the amine nitrogen of 0.4%and 0.5%. The rate of solids equal to 7%. 9-10 day increase of this index is terminated, racks off, hydrolyzed assert within 2 days. Then filtered through filter cloth. The filtrate add 1% chloroform, proscout rubber tube, stored at a temperature of from 2 to 8°C in a cold chamber. Use as needed.

Nutrient medium for enzymatic basis l is Gogo 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 other ingredients in the ratio, g/l: potassium phosphate substituted 1 - 0,9; potassium phosphate 2-substituted 3-water - 2,2; activated charcoal - 2,0; microbiological agar - 8,5; distilled water to 1 L. the Mixture is brought to a boil and boil until complete melting of agar for 2 min, pH adjusting solution of hydrochloric acid (1:1) in an amount of 5 ml to pH 6,8±0,1. Prepared nutrient medium is poured into a graduated vials of 200±0.5 ml, which is hermetically sealed cotton-gauze tubes and paper Craft. Sterilized at 1 ATM for 15 minutes In melted in a boiling water bath and cooled to 56°C agar add L-cysteine hydrochloride 0.35 g, enzymatic hydrolyzed peanut 10.0 ml, then poured into sterile Petri dishes. Dry for 30 minutes and used for sowing.

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, 2007), using as the test cultures Legionella Legionella pneumophilla ADS 33155 Bloomington, Legionella pneumophilla ATCC 33152 Philadelphia.

Subjects cult of the s were grown on plates of solid agar control environment SAL pH of 6.8 at 37°C 24 h From overnight cultures were prepared 1 billion suspension M.L. test strains equal to 10 units of optical turbidity standard of CCA gisk named after. L.A. Lytvyn, then serial tenfold dilutions in physiological solution of 4.5 ml conveyed to the content in 1 ml of 1000 (10-6and 100 M.K. (10-7). From these breeding suspension cultures were sown in 0.1 ml of 3 Petri dishes spilled dried test agar, on Wednesday SAL and agar of Hottinger. Crops were incubated at 37°C for 5 days. Through 24-120 h took into account the results by counting the grown colonies.

Control environment the SAL (Nutrient medium for the cultivation and selection of the causative agent of legionellosis elective) - composition g/l: nutrient basis of animal origin - fermentolizat spleen liquid cattle in the dry substance - 7,0; activated carbon shelter-But 4.0; agar microbiological 17,0; potassium phosphate one-deputizing 1,6; potassium phosphate disubstituted trihydrate 3,4; pH environment of 7.1 (Manufacturer FGUZ Growth NPCI of Rospotrebnadzor. 344002, Rostov-on-don, street M. Gorky, 117).

Control of agar Hottinger. Industrial regulation No. 1707-05 for the production of nutrient agar for cultivation of microorganisms, ready-to-use (agar of Hottinger). Registration certificate № SDF 2009/05571 dated August 20, 2009 for a Term not exhaust unicen.

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 mg% - 240,0 ml; potassium phosphate 1 - substituted - 0,7; potassium phosphate 2-substituted 3-water - 2,0; activated charcoal - 1,0; L-cysteine hydrochloride - 0,25; enzymatic hydrolyzed peanut - 8,0; microbiological agar - 8,0; distilled water to 1 L. this ratio of ingredients was increased by an average of 22 and 2 convex, bluish-grayish colonies with a diameter of 1.5 mm after 48 h for both strains, taken in experience, whereas the control environment, the SAL growth was observed only after 72 h in the amount of 19 and 2, respectively, and on agar of Hottinger growth was absent.

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 0,120 mg% - 260,0 ml; potassium phosphate 1 - substituted - 0,9; potassium phosphate 2-substituted 3-water - 2,2; activated charcoal - 2,0; L-cysteine hydrochloride - 0,35; enzymatic hydrolysate of peanuts - 10, microbiological agar - 10,0; distilled water to 1 L. this ratio of ingredients was increased 86 and 8 PLANO-convex, bluish-grayish colonies with a diameter of 1.5 mm through 48 h for both a TEC is-strains taken in experience, whereas the control environment, the SAL growth was observed only after 72 h at 60 and 6, respectively, and on agar of Hottinger growth was absent.

Example 3. 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 0,140 mg% - 280,0 ml; potassium phosphate 1-substituted - 1,1; potassium phosphate 2-substituted 3-water - 2,4; activated - 3,0; L-cysteine hydrochloride - 0,45; enzymatic hydrolyzed peanut - 12,0; microbiological agar - 12, 0mm; distilled water to 1 L. this ratio of ingredients was increased by 15 and 5 convex, bluish-grayish colonies with a diameter of 1.5 mm after 48 h for both test strains, taken in experience, whereas the control environment, the SAL growth was observed only after 72 hours of 11 and 1, and on agar of Hottinger growth was absent.

The obtained results allowed to determine the optimal variant of the nutrient medium on the basis of enzymatic hydrolysate of pig lung (example 2).

Nutrient media for cultivation of Legionella containing a nutrient basis, potassium phosphate 1-substituted; potassium phosphate 2-substituted 3-water; activated carbon; L-cysteine hydrochloride; agar microbiological; distilled water, characterized in that it is omnitele contains as a stimulant of the enzymatic hydrolysate of peanuts, and as a nutrition - enzymatic hydrolysate easy pigs in the following ratio of ingredients, g/l:

enzymatic hydrolysate of the lung of a pig260,0
potassium phosphate 1-substituted0,9
potassium phosphate 2-substituted
3-water2,2
activated carbon2,0
L-cysteine hydrochloride0,35
enzymatic hydrolysate of peanuts10,0
agar Microbiology10,0
distilled waterto 1 l



 

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