RU2380409C1 - Nutrient medium for pestilential microbe cultivation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: culture medium for pestilential microbe cultivation contains in g/l: enzymic potato hydrolyzate 310.0-410.0 as a base of the nutrient medium, sodium chloride 4.0-6.0, two-substituted sodium phosphate 3.0-5.0, sodium sulphite 0.3-0.5, microbiologic agar 10.0-14.0, and distilled water the rest.

EFFECT: optimal conditions for the growth of a pestilential microbe ensured by improved quality and transparency of the protein nutrient medium.

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Description

The invention relates to biotechnology, namely to obtain nutrient media that create optimal conditions for the cultivation of plague microbe.

A nutrient medium for cultivating a plague microbe is known, for the preparation of which peeled, finely chopped potatoes are poured with a double amount of tap water, heated at 133 ° C for 10 minutes, left to cool for 15 minutes to cool, then the upper layer of liquid is drained and added to it 0.5% sodium chloride and 1% peptone or Hottinger digest (Culture media in medical microbiology. Medgiz. 1950. Kozlov Yu.A. 1950. p.106-107).

The disadvantage of this environment is its opacity.

Closest to the invention is patent RU 2260620 C2 (publ. Bull. No. 26 09/20/2005), the combination of features of which is closest to the set of essential features of the invention, which describes a nutrient medium for cultivation of the plague microbe, including, g / l: as a nutrient base, the enzymatic hydrolyzate of soybeans - 320-420, sodium chloride - 4.0-6.0, sodium disubstituted phosphate - 3.0-5.0, sodium sulfite - 0.3-0.5, lipoic acid - 0 , 4-0.6, 20% sodium hydroxide solution 2.5-3.5 and distilled water up to 1 liter.

The aim of the invention is to obtain high-quality transparent nutrient medium for the cultivation of plague microbe on a protein basis only from plant materials - potatoes.

The essence of the invention lies in the fact that the nutrient medium as an nutrient base contains an enzymatic hydrolyzate of potato and a stimulating additive sodium sulfite in the following ingredients, g / l:

Enzymatic Potato Hydrolyzate 310.0-410.0 Sodium Chloride 4.0-6.0 Disubstituted sodium phosphate 3.0-5.0 Sodium Sulphate 0.3-0.5 Microbiological agar 10.0-14.0 Distilled water up to 1 l

To prepare a nutrient base, potatoes containing an average of 2% nitrogenous substances are used as feedstock, in addition, potatoes are rich in trace elements and vitamins.

As a rule, plant foods, unlike animals, contain more carbohydrates than proteins and fats. The exception is protein-rich plants. So potato contains an average of g / l: protein 2.6; glucose 5.41; potassium 0.7; sodium 1.01; calcium 0.02; iron 2.51, water-soluble vitamins (Kozlov Yu.A., 1950). Amino acids are formed from protein substances in potatoes during hydrolysis: lysine, methionine, tryptophan, cystine, threonine, serine, alanine, glycine, valine, leucine, tyrosine, histidine, which differ little in quantity and quality from amino acids of meat hydrolysates. Potato tubers contain 12–20% starch, which is a mixture of amylose 15–25% (with a molecular weight of 100,000 to 600,000) and amylopectin 75–85% (shell material with a molecular weight of about 1,000,000). In addition, potato proteins are characterized by an increased content of carbohydrates, which are closely related to carbohydrate metabolism through a cycle of tricarboxylic acids that supply macroergic bonds (Y.P. Ypta, 1987).

Preparation of the nutrient basis of the proposed nutrient medium for growing microorganisms is as follows.

Take 1.0 ± 0.05 kg of potato tubers, wash it with a brush in running water, peel it, cut it into 2 × 2 cm slices, pour it into an enameled pan, pour 3 l of drinking water, boil for 5 minutes, cool up to 46 ° C. A decoction of potatoes is poured into a five-liter glass bottle. The cattle pancreas, passed through a meat grinder twice at the rate of 100 g / l, is added to a bottle with a potato broth. Set the pH to 8.2 ± 0.1 phenolphthalein to a slightly pink color with sodium carbonate in an amount of 5.0 g / L. Chloroform is added as a preservative in an amount of 1% to the volume of liquid. The cylinder is closed with a cotton-gauze stopper, parchment on top, mixed thoroughly and placed in a heat chamber at a temperature of 37 ± 1 ° С. The contents of the balloon during the day are stirred every 20 ± 1 min, the next day - every 2 hours.

The dynamics of the hydrolysis process is monitored daily by determining amine nitrogen. The end of hydrolysis is judged by the achievement of amino nitrogen of 0.3% -0.4%. After this, heating and stirring are stopped, the hydrolyzate is left to stand for two days. The settled upper layer of the hydrolyzate is decanted with a rubber hose and filtered through a linen filter. The filtrate is collected in a glass container with a capacity of 5 l, chloroform is added in an amount of 1% to the volume of the container for preservation, closed with a sterile rubber stopper and transported to a cold chamber where it is stored at a temperature of +2 to + 8 ° С.

A nutrient medium based on a potato enzymatic hydrolyzate for growing microorganisms is prepared in the following way. Take an enzymatic hydrolyzate from potatoes diluted with distilled water to an amine nitrogen reading of 0.12% -360.0 ml; sodium chloride - 5.0 g; sodium phosphate disubstituted - 4.0 g. For clarification of the obtained liquid, activated carbon of the UO-A brand is used in an amount of 2% by volume of the medium, which is poured into an enameled pan, distilled water is added - up to 1 liter, the medium is thoroughly mixed until a uniform masses. Heated to 56 ° C, then set the pH to 8.2 ± 0.1 using a 20% solution of sodium hydroxide in an amount of 8.0 ml, boiled for 5 minutes Filter through a cloth and 8-16 layers of filter paper, collecting the filtrate is carried out in an enameled pan. Microbiological agar, 12 g / l, is added to the ready-made light yellow filtrate. The mixture was brought to 100 ° C and boiled until the agar was completely melted for 10 minutes. The pH is adjusted with a solution of hydrochloric acid (1: 1) to a pH of 7.2 ± 0.1, sodium sulfite is added at a concentration of 0.4 g / L.

The prepared medium is poured into graduated bottles of 200 ± 0.5 ml, which are hermetically sealed with cotton gauze plugs and Kraft paper. Sterilized at 0.5 atm for 30 minutes. After melting in a boiling bath, the sterile medium is poured into sterile Petri dishes, dried for 30 minutes and used for inoculation.

As an example, cultures of the plague microbe vaccine strain EB and virulent No. C-802 were tested; S-803; 461; S-740; C-739 grown on plates of 2% Hottinger agar, pH 7.2 at 28 ° C for 24 hours. From daily agar cultures of plague microbe test strains, a suspension is prepared in a 0.9% sodium chloride solution with a density of 1.0 · 10 ⁹ MK./ml according to the standard sample turbidity GISK them. L.A. Tarasovich OSO 422859-86 P-10 units and do 10-fold serial dilutions, transferring 0.5 ml of suspension in 4.5 ml of 0.9% sodium chloride solution to a dilution of 10 ^-7 (1.10 ² m.k. ./ml), mixing thoroughly, changing the sterile pipette after each dilution. A culture from a dilution of 10 ^-6 (1000 m.k. / ml) and 10 ^-7 (100 m.k. / ml) is applied 0.1 ml per 3 freshly prepared and well-dried agar plates. The seeded culture is distributed over the surface of the agar by shaking the Petri dish. The results of the culture growth rate in each dilution of the microbial suspension are taken into account after 48 hours of incubation at 28 ° C.

The rate of growth rate is determined by the minimum incubation time of crops, during which, with appropriate dilution, a clearly visible growth of the culture is ensured in all seeded cups with a dense medium and the formation of typical colonies on plates with dense medium that are easily differentiable by microscopy.

Assessment of the growth properties of the nutrient medium for cultivation of the plague microbe is carried out by counting the number of grown colonies on agar plates.

Example 1

Test strains were grown on a nutrient medium containing, g / l: enzymatic potato hydrolyzate - 310.0; sodium chloride - 4.0; disubstituted sodium phosphate - 3.0; sodium sulfite - 0.3; microbiological agar - 10.0; distilled water - up to 1 liter.

With this ratio of ingredients, a typical growth of the colonies grown on agar plates was observed for the plague microbe at a sowing dose of 100 and 10 mk. after 48 h, respectively, 50 and 5 with a diameter of 1.0 mm.

Example 2

Test strains were grown on a nutrient medium containing g / l: enzymatic potato hydrolyzate - 360.0; sodium chloride - 5.0; disubstituted sodium phosphate - 4.0; sodium sulfite - 0.4; microbiological agar - 12.0; distilled water - up to 1 liter.

With this ratio of ingredients, a typical growth of the colonies grown on agar plates was observed for the plague microbe at a sowing dose of 100 and 10 mk. after 48 h, respectively 57 and 6 with a diameter of 1.5 mm.

Example 3 Test strains were grown on a nutrient medium containing, g / l: enzymatic potato hydrolyzate - 410.0; sodium chloride - 6.0; disubstituted sodium phosphate - 5.0; sodium sulfite - 0.5; microbiological agar - 14.0; distilled water - up to 1 liter.

With this ratio of ingredients, a typical growth of plague microbe colonies was observed at a sowing dose of 100 and 10 mk. after 48 h, respectively, 50 and 5 with a diameter of 1.0 mm.

Thus, the claimed nutrient medium for cultivation of the plague microbe (example 2), prepared on the basis of an enzymatic hydrolyzate of potato, may well replace the nutrient medium based on beef meat.

Claims (1)

Nutrient medium for cultivation of the plague microbe, containing a nutrient base, sodium chloride, sodium phosphoric acid disubstituted, sodium sulfate and distilled water, characterized in that it contains an enzymatic hydrolyzate of potato and microbiological agar in the following ratio of ingredients as a nutrient base, g / l:
potato enzymatic hydrolyzate 310.0-410.0 sodium chloride 4.0-6.0 disubstituted sodium phosphoric acid 3.0-5.0 sodium sulfide 0.3-0.5 microbiological agar 10.0-14.0 distilled water up to 1 l