RU2795907C1 - Selective nutrient medium for isolating aeromonads from water bodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient selective medium for the isolation of aeromonads from water bodies, which contains peptone, fodder yeast extract, arginine, monosubstituted potassium phosphate, magnesium sulfate, sodium chloride, deoxycholate, sodium hydroxide, bromthymol blue, thymol blue, ampicillin and distilled water.

EFFECT: improving the accumulative and selective properties for separating aeromonads from water bodies.

1 cl, 5 tbl

Description

The invention relates to the field of microbiology, more specifically to the field of sanitary microbiology and can be used by institutions that supervise and control the quality of waste water, drinking water and surface water, including those of fishery importance.

A known method for the isolation and identification of aeromonads using nutrient media A1 and A2, in accordance with the recommendations "Methods for the study of environmental objects and pathological material for aeromonads" (Methods for the study of environmental objects and pathological material for aeromonads: Method. Recommendations / Ministry of Health RSFSR, Chief Directorate of Scientific Research Institute and Coordination of Scientific Research, [compiled by G. P. Kalina, T. I. Grafova], Moscow: B. I., 1980. - 11 p. ), developed at the Moscow Research Institute of Hygiene. F.F. Erisman;

Liquid accumulation medium A-1 has the following composition (g/l):

Magnesium sulfate 0.2 Starch soluble 2 Gelatin 10 Potassium hydrogen phosphate 1 Aqueous solution of crystal violet 0.01% 20 cm ³ pH 7.2 - 7.4 Sterilization 112 +/- 1 for 15 min

The selectivity of medium A-1 is due to the action of crystal violet in relation to most gram-positive bacteria.

Known nutrient medium for the detection of aeromonads by copyright certificate SU No. 544674 dated 24.07.1975 for the invention. Nutrient medium for the detection of aeromonads in environmental objects, containing starch and distilled water, and in order to accelerate the process of growing aeromonads, it additionally contains sodium ammonium phosphate, potassium phosphate monosubstituted, magnesium sulfate, crystal violet and all ingredients are taken in the following ratios, weight . %:

Sodium ammonium phosphate 0.1-0.2 Potassium phosphate monosubstituted 0.05-0.1 Magnesium sulfate 0.01-0.03 Starch 0.5-1.5 Crystal violet 0.0002-0.00025 Distilled water Rest

When growing aeromonads in this medium, the growth of extraneous microflora is almost completely absent. The disadvantage of the medium for the isolation of aeromonads is the presence in the composition of only one source of nutrients (starch), in addition, there is a weak suppression of the growth of enterobacters and enterococci, and with high fecal water pollution, the growth of Escherichia coli can be observed.

The objective of the claimed invention was to develop a liquid nutrient medium for the isolation of aeromonads from water, which could ensure sufficient growth of bacteria of the genus Aeromonas while completely suppressing Escherichia, Salmonella, Staphylococcus and Enterococcus. The technical result is to increase the storage and selective properties of the environment for the selection of aeromonads from water bodies. This technical result is achieved through the totality of the proposed components of the environment.

The essence of the invention lies in the fact that Nutrient selective medium for isolating aeromonads from water bodies contains peptone, fodder yeast extract, arginine, monosubstituted potassium phosphate, magnesium sulfate, sodium chloride, deoxycholate, sodium hydroxide, bromthymol blue, thymol blue, ampicillin and distilled water in the following ratio of components, g/ l:

Peptone enzymatic 10 fodder yeast extract 3 L-arginine hydrochloric acid 3.5 sodium chloride 9 Potassium phosphate monosubstituted 1 Magnesium sulfate 0.2 Sodium deoxycholate 2 Sodium hydroxide 0.35±0.05 Bromothymol blue 0.03 thymol blue 0.03 Ampicillin 0.005 Distilled water up to 1000 ml

Bacteria of the genusAeromonas belong to the Aeromonadaceae family. These are facultative anaerobic gram-negative rods, more often mobile, oxidase-positive and catalase-positive. According to the fermentation of carbohydrates, the formation of hydrogen sulfide and the hydrolysis of urea, a variation of signs is noted. Under natural conditions, they are able to multiply at temperatures from 4°C to 45°C, as well as at a pH of 4.5 to 9.8. Aeromonads are widely distributed in fresh and salt water bodies, can be found in sewage, bottom sediments and in the microflora of aquatic organisms. Among them there are species pathogenic for marine, freshwater fish, amphibians and reptiles. Since fishery reservoirs are often located near settlements and agricultural enterprises, they receive various effluents (municipal, livestock, etc.), which leads to bacterial pollution of water, which results in epizootic trouble of water bodies. Some of the aeromonad species can initiate human diseases associated with both water and nutritional factors. Aeromonads are known as microorganisms that form extensive colonies in water systems. These bacteria have been reported to be found in treated tap water distribution systems and biofilms in distribution systems where they may be resistant to chlorination. In connection with the foregoing, water analysis is carried out, including the indication and quantitative accounting of bacteria of the genusAeromonas. Peptone is used as the main source of nitrogen in the proposed medium, fodder yeast extract and L-arginine hydrochloric acid are used as growth factors, sodium chloride is used as a phosphate buffer, potassium phosphate monosubstituted as a phosphate buffer, and sulfate is a cofactor for various metabolic reactions. magnesium, selective factors are sodium deoxycholate and ampicillin, indicators are bromthymol blue, thymol blue and distilled water in the following ratio of components, g/l:

Peptone enzymatic 10 g 34.35% fodder yeast extract 3 g 10.30% L-arginine hydrochloric acid 3.5 g 12.02% sodium chloride 9 g 30.91% Potassium phosphate monosubstituted 1 g 3.43% Magnesium sulfate 0.2 g 0.69% Sodium deoxycholate 2 g 6.87% Sodium hydroxide 0.35±0.05 g 1.21% Bromothymol blue 0.03 g 0.10% thymol blue 0.03 g 0.10% Ampicillin 0.005 g 0.02% Distilled water up to 1000 ml pH 8.3±0.2 at 25°C

The introduction of sodium deoxycholate into the nutrient medium promotes the inhibition of gram-positive bacteria. The addition of ampicillin completely inhibits the growth of Salmonella, Escherichia coli and enterococci sensitive to it, thereby creating favorable conditions for the growth of aeromonads. The high alkalinity of the medium (pH 8.3 ± 0.2) helps to suppress the growth of enterobacteria and Pseudomonas aeruginosa. Under the action of aeromonad arginine dehydrolase on L-arginine of the medium, alkaline reaction products are formed, which change the color of the medium from the initial green to blue.

Selection of the concentrations of ingredients in the composition of the proposed nutrient medium for aeromonads, using different concentrations of peptone, fodder yeast extract, L-arginine hydrochloric acid, sodium deoxycholate, ampicillin and indicators (table 2).

table 2 The composition of the nutrient medium in g/l: 1 option Option 2 3 option Peptone enzymatic 5.0 10.0 10.0 fodder yeast extract 1.0 3.0 3.0 L-arginine hydrochloric acid 2.0 3.5 3.5 sodium chloride 9.0 9.0 9.0 Potassium phosphate monosubstituted 1.0 1.0 1.0 Magnesium sulfate 0.2 0.2 0.2 Sodium deoxycholate 3.0 2.5 2.0 Sodium hydroxide 0.22±0.05 0.35±0.05 0.35±0.05 Bromothymol blue 0.03 0.03 0.03 thymol blue - - 0.03 Ampicillin - - 0.005 pH at 25°C 7.4±0.2 8.3±0.2 8.3±0.2

When selecting the concentrations of ingredients used test strains: Pseudomonas aeruginosa 10145; Salmonella paratyphi B 506; E. coli 3912/41; Staphylococcus aureus 6538; Enterococcus faecalis 29212, Aeromonas caviae VKShM-B-297M; Aeromonas veronii VKSHM-B-307M; Aeromonas hydrophila VKShM-B-301M, Aeromonas salmonicida VKShM-B-307M. To prepare microbial suspensions, daily agar cultures of test strains are washed off with 0.9% sterile isotonic sodium chloride solution, suspensions are adjusted to 10 units. according to the optical standard of turbidity, which corresponds to 10 ⁹ microbial cells in 1 ml. Then serial tenfold dilutions are brought to a content of 100 thousand (10 ^-4 ), 10 thousand (10 ^-5 ), 1 thousand (10 ^-6 ), 100 (10 ^-7 ), 10 (10 ^-8 ) and 1 ( 10 ^-9 ) microbial cells in 1 ml of suspension. Seeding is performed from each dilution of 1 ml in 9 ml of the experimental medium (media options 1-3) in triplicate and incubated at a temperature of 28±2°C for 18-24 hours. Inoculation is carried out from test tubes with variants of experimental media in which bacterial growth was observed, onto plates with nutrient media (fish-peptone agar, Endo agar, blood agar).

Data on the growth properties of the medium are presented in tables 3 and 4.

Table 3 Dependence of the growth of test strains of microorganisms on the concentration of ingredients in the experimental environment Microorganisms Breeding Experimental environment options 1 2 3 P. aeruginosa 10 ^-1 + + + ^10-2 + - - ^10-3 - - - ^10-4 - - - S. paratyphi B 10 ^-1 + + - ^10-2 + + - ^10-3 + - - ^10-4 + - - E. coli 10 ^-1 + + - ^10-2 + + - ^10-3 + - - ^10-4 - - - S. aureus 10 ^-1 - - - ^10-2 - - - ^10-3 - - - ^10-4 - - - E. faecalis 10 ^-1 + + - ^10-2 - - - ^10-3 - - - ^10-4 - - -

Table 4 Dependence of the growth of aeromonads on the concentration of ingredients in the experimental environment Microorganisms Breeding Experimental environment options 1 2 3 A. hydrophila ^10-6 + + + ^10-7 - + + ^10-8 - + + ^10-9 - - - A. veronii ^10-6 + + + ^10-7 + + + ^10-8 - + + ^10-9 - - + A. salmonicida ^10-6 + + + ^10-7 + + + ^10-8 - + + ^10-9 - - - A. caviae ^10-6 + + + ^10-7 - + + ^10-8 - + + ^10-9 - - -

The growth of bacteria of the genus Aeromonas in an amount corresponding to the inoculum dose of the microorganism was noted on a medium with a minimum and optimal content of the nutrient base (enzymatic peptone, fodder yeast extract, L-arginine). The predominant growth of aeromonads was recorded at the optimal content of the nutrient base. At the same time, the visible effect of changing the color of the medium from the initial green to blue was manifested with an increase in the content of L-arginine from 2.0 g to 3.5 g. aeromonad is not reached. In the process of growth, Pseudomonas aeruginosa does not change the initial green color of the medium. The growth of other microorganisms ( E. coli, S. paratyphi B, S. aureus, E. faecalis ) depends on the inoculum dose of the microorganism and no or significant growth inhibition was observed on the medium with sodium deoxycholate and ampicillin. Increasing the amount to 3 g of sodium deoxycholate does not affect the growth of enterococci and Staphylococcus aureus, compared with the indicated parameters. At the same time, reducing the amount of inhibitory substance reduces the cost of the medium. The nutrient medium has good growth and accumulation properties, the accumulation efficiency is an order of magnitude higher than in the GRM-broth (table 5).

Table 5 Comparison of the sensitivity of nutrient accumulation media for the isolation of aeromonads from water bodies Test strains Experimental environment timing broth Breeding Breeding ^{10-7 10-8 10-9 10-7 10-8 10-9} A. hydrophila + + - + - - A. veronii + + + + + - A. salmonicida + + - + - - A.caviae + + - + - -

The method of preparing a nutrient medium for isolating aeromonads is as follows: in a chemically clean glass heat-resistant flask, for example, a two-liter round flat-bottomed or conical flask according to GOST 25336-82, weighings are added in g/l: enzymatic peptone - 10.0; sodium chloride - 9.0; fodder yeast extract - 3.0; potassium phosphate monosubstituted 1.0 g (in accordance with GOST 4198-73 NDA); magnesium sulfate - 0.2 g and distilled water is added to 1 liter, followed by stirring until complete dissolution. Then the resulting mixture is heated, followed by stirring until they are completely dissolved. After that, weights are added in g/l: L-arginine hydrochloric acid (in accordance with TU 6-09-2100-78) - 3.5 g, an alcohol solution of bromthymol blue water-soluble (in accordance with TU 6-09-07-1602- 87) - 0.03 g (in the form of a 1.6% alcohol solution - 1.88 ml), thymol blue (in accordance with TU 6-09-3501-78) - 0.03 g and sodium hydroxide (in accordance with GOST 4328-77 NDA) - 0.35 ± 0.05 g (in accordance with GOST 4328-77 KhCh). Next, adjust the pH to 8.3±0.2 using 20% aqueous sodium hydroxide solution. Then sterilized by autoclaving at 1.1 atm. (121°C) for 10 min. Then 2.0 g of sodium deoxycholate and 0.005 g of ampicillin are added to the cooled medium. It is poured into sterile vials and test tubes and cultivated at a temperature of 28 ± 2°C for 18-24 hours.

Claims (2)

Nutrient selective medium for the isolation of aeromonads from water bodies, containing peptone, fodder yeast extract, arginine, monosubstituted potassium phosphate, magnesium sulfate, sodium chloride, deoxycholate, sodium hydroxide, bromthymol blue, thymol blue, ampicillin and distilled water in the following ratio of components, g /l: Peptone enzymatic 10 fodder yeast extract 3 L-arginine hydrochloric acid 3.5 sodium chloride 9 Potassium phosphate monosubstituted 1 Magnesium sulfate 0.2 Sodium deoxycholate 2 Sodium hydroxide 0.35±0.05 Bromothymol blue 0.03 thymol blue 0.03 Ampicillin 0.005 Distilled water up to 1000 ml