RU2484141C1 - Elective-differential nutrient medium for extraction of choleraic vibrios - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention is an elective nutrient medium, which may be used in laboratory practice for extraction of a cholera agent. The nutrient medium contains a nutrient base, microbiological agar, saccharose, sodium chloride, sodium carbonate, bismuth nitrate pentahydrate, sodium citrate, cattle bile, EDTA iron (III)-sodium salt, bromthymol blue, mesoinositol, activated charcoal, sodium sulfite and L-cysteine, potassium tellurite and distilled water. The nutrient base in the nutrient medium is a dry nutrient broth from a pancreatic overcook of Caspian sprat or a dry nutrient broth from hydrolysate of fish and bone flour or a complex of yeast extract and peptone at the specified ratio of components.

EFFECT: invention makes it possible to increase yield of choleraic vibrio and to increase differentiating properties of the medium.

11 tbl, 11 ex

Description

The present invention relates to the field of medical microbiology and is an elective nutrient medium that can be used in laboratory practice to isolate the causative agent of cholera.

Known medium for the selective selection of cholera vibrio TCBS (1) (analog), including, g / l: yeast extract - 5.0; peptone - 10.0; sodium thiosulfate - 10.0; sodium citrate - 10.0; bile bovine - 8.0; sucrose - 20.0; sodium chloride - 10.0; iron citrate - 1.0; bromothymolblow - 0.042; timolblau - 0.04; agar - 14.0; distilled water - 1 liter at a pH of 8.6.

A disadvantage of the known medium is its high cost and low selectivity with increased load of seed.

Closest to the proposed nutrient medium is an electrically differential nutrient medium (SEDH) for the isolation of cholera vibrios (prototype) developed at the Rostov-on-Don Antiplague Institute (State registration of medicines and medical devices No. 93/270/17, Temporary pharmacopoeial article VFS 42-41, BC - 93 (2)), containing, g / l: peptone D - 6.0; microbiological agar - 10.0; sodium carbonate - 1.0; sodium chloride - 8.0; Detergent "Progress" according to TU 36-10719-77 - 6.0; potassium telluric acid - 0.03; bromothymol blue - 0.04; sucrose - 20.0; distilled water - 1 liter at a pH of 8.6.

However, the industrial production of the nutrient base of the medium - peptone D (a digest of hydrocarbon-oxidizing yeast) was discontinued, and the Progress preparation underwent a technological modification, as a result of which it significantly lost its activity, and the medium as a whole decreased its inhibitory properties with respect to commensal microorganisms, in particular , to test microbes - Proteus and Escherichia coli (inhibited the growth of both microbes only at a concentration of 10 ⁵ mk). Due to the need to monitor cholera vibrios, the need for an elective environment that allows you to select cholera vibrio from a person and the environment remains high.

The technical task of the invention is the creation of a new electrically differential environment SEDH-M (modernized) to isolate cholera vibrios with increased ability to inhibit the accompanying extraneous microflora.

The problem is achieved by the fact that in the known elective-differential nutrient medium for the selection of cholera vibrios,

containing a nutrient base, microbiological agar, sucrose, sodium chloride, sodium carbonate, potassium tellurite, bromothymol blue and distilled water, bismuth nitrate pentahydrate, sodium citrate, cattle bile, EDTA iron (III) - sodium are additionally used as inhibitors of extraneous microflora in the medium salt, bromothymol blue is introduced in a larger quantity, and meso-inositol, activated carbon, sodium sulfite and L-cysteine are introduced to stimulate the growth of cholera vibrio, while the nutrient base is a dry nutrient minutes pancreatic digest broth Caspian sprat the following component ratio, g / l:

Pancreatic Dry Nutrient Broth Caspian sprat 8.0-12.0 Microbiological agar 8.0-12.0 Sucrose 18.0-22.0 Sodium chloride 6.0-10.0 Sodium carbonate 0.7-0.9 Sodium Citrate 8.0-12.0 Sodium sulfite 0.2-0.4 Bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 Activated carbon 1.0-1.4 L-cysteine 0.2-0.4 Potassium tellurite 0.001-0.003 Bismuth pentahydrate 0.06-0.1 Cattle bile 2.0-4.0 Distilled water 1 liter

at pH 8.0.

At the same time, in the elective differential nutrient medium for the isolation of cholera vibrios, the dry nutrient broth from the fish-bone meal hydrolyzate was used as the basis in the following ratio of components, g / l:

Hydrolyzed Dry Nutrient Broth fish bone meal 8.0-12.0 Microbiological agar 8.0-12.0 Sucrose 18.0-22.0 Sodium chloride 6.0-10.0 Sodium carbonate 0.7-0.9 Sodium Citrate 8.0-12.0 Sodium sulfite 0.2-0.4 Bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 Activated carbon 1.0-1.4 L-cysteine 0.2-0.4 Potassium tellurite 0.001-0.003 Bismuth pentahydrate 0.06-0.1 Distilled water 1 liter

at pH 8.0.

In addition, in the elective-differential nutrient medium for the isolation of cholera vibrios, the complex — yeast extract and peptone — was used as the nutrient base in the following ratio of components, g / l:

Yeast extract 5,0 Peptone 10.0 Microbiological agar 8.0-12.0 Sucrose 18.0-22.0 Sodium chloride 6.0-10.0 Sodium carbonate 0.7-0.9 Sodium Citrate 8.0-12.0 Sodium sulfite 0.2-0.4 Bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 Activated carbon 1.0-1.4 L-cysteine 0.2-0.4 Potassium tellurite 0.001-0.003 Bismuth pentahydrate 0.06-0.1 Cattle bile 2.0-4.0 Distilled water 1 liter

at pH 8.0.

The nutrient medium is prepared as follows.

As a nutrient base, take dry nutrient broth produced by NPO "Nutrient media", Makhachkala (Russia), (see M. Medzhidov "Guide to microbiological nutrient media", M. Medicine, 2003, p.24), prepared on the basis of pancreatic digest of Caspian sprat, or other suitable nutritional bases indicated below (options).

The following is successively introduced into the container: dry nutritional broth 8.0-12.0; microbiological agar 8.0-12.0; sucrose 18.0-22.0; sodium chloride 6.0-10.0; sodium carbonate 0.7-0.9; sodium citrate 8.0-12.0; sodium sulfite 0.2-0.4; bromothymol blue 0.6-0.1.0; mesoinositis 0.6-1.0; EDTA iron (III) sodium salt 0.4-0.6; activated carbon 1.0-1.4; L-cysteine 0.2-0.4; potassium tellurite 0.001-0.003; bismuth penta nitrate 0.06-0.1 and add 1 liter of cold distilled water. The mixture is boiled in a steam sterilizer for 20 minutes at 100 ° C with thorough stirring. Then the liquid is cooled and bile of cattle 2.0-4.0 is introduced. The solution is filtered through a cotton-gauze filter. The reaction of the medium is checked and the pH is adjusted to 8.0 using an 18% hydrochloric acid solution or a 20% sodium hydroxide solution.

The finished medium, having a dark purple color, is poured into bottles or Petri dishes. The medium does not need sterilization.

Vibrio cholerae on SEDH-M medium one day after incubation at 25-40 ° C grow in the form of smooth, shiny, flat-convex, red-orange colonies, sometimes with a dark brown center and a narrow rim of the transparent bluish periphery. When a needle or loop is immersed and removed from the colony, a short cord (semi-viscosity) is formed. For cholera colonies, a dull yellowish color is not characteristic, a liquidish consistency without a strand. When working with the SEDH-M medium, a stereo microscope with oblique overhead illumination (magnification - 12.5 × 2) is useful for searching for suspicious colonies and pinning material from them.

On SEDH and TCBS media, vibrio colonies are homogeneous, convex, smooth, and yellow.

The proposed SEDH-M medium was subjected to a series of laboratory and field tests, during which its growth and elective properties were tested, and in model experiments, the ability to isolate cholera vibrios from artificially contaminated stool samples and water samples from natural sources was tested.

Data on the growth and elective properties of the SEDH-M medium prepared on a nutritious basis from the pancreatic digest of Caspian sprats with various concentrations of this and other ingredients (examples 1, 2, 3, 4, 5) were obtained using cholera test strains: Vibrio cholerae O-1 P-1 (145) classic biovar, V. cholerae O-1 M-878 biovar eltor and commensal Escherichia coli 18 Escherichia coli strains and Proteus vulgaris HS 19 N 222. The comparison medium was the prototype SEDC . In a similar way, the growth and elective qualities of the SEDH-M medium prepared using two other nutrient bases — dry nutritious broth from the fish and bone meal hydrolyzate of the GRM fish production of the SSC Obolensk, Russia, and a combination of yeast extract with peptone manufactured by Merck, Germany, were tested. (examples 6, 7, respectively).

In model experiments, in comparison with the prototype SADC medium and the TCBS analogue medium, the growth properties of the SEDC-M medium in relation to seven field strains of cholera vibrios — V. cholerae O139 17918 ctx–; V. cholerae O139 16064 ctx +; V. cholerae O1 26 ctx-; V. cholerae O1 24 ctx-; V. cholerae O1 12278 ctx +; V. cholerae O1 43 ctx-; V. cholerae no O1 16147 ctx - from the Rostov NIEPCH collection (Example 8), - we studied the probability of V. cholerae isolation on all three media from samples of stools of a healthy person (Example 9) and from samples of natural river water that did not initially contain vibrios (example 10) and artificially contaminated with the indicated seven field strains. In conclusion of the tests, the results of surveys using the SEDH-M medium of water samples of the Don River for cholera vibrios carried out as part of the monitoring for cholera in 2010 / (example 11) are presented.

Example 1

To prepare the environment SEDH-M use the ingredients in the following minimum concentrations, g / l: dry nutrient broth from pancreatic digest of Caspian sprat - 6.0; microbiological agar - 6.0; sucrose - 16.0; sodium chloride - 4.0; sodium carbonate - 0.6; sodium citrate - 6.0; sodium sulfite - 0.1; bromothymol blue - 0.5; mesoinositis - 0.4; EDTA iron (III) sodium salt - 0.3; activated carbon - 0.8; L-cysteine - 0.1; potassium tellurite - 0.0005; bismuth pentahydrate nitrate - 0.04; cattle bile - 1.0.

These reagents are poured into 1.0 l of distilled water, and dissolved with stirring and boiling for 20-30 minutes. The reaction medium is adjusted to 8.0. The finished medium is poured into Petri dishes. Similarly, a prototype SEDH medium was prepared in the original recipe. Sowing of two test cultures of cholera vibrio and two test cultures of commensal microflora and incubation of cups with crops were carried out according to the generally accepted methodology (3, 4).

The results characterizing the growth and inhibitory properties of the environment SEDH-M prepared according to this recipe, and the environment of the prototype SEDH are shown in table 1.

The table shows that both media with a satisfactory growth effect (from 100 seeded m.k. strains of Escherichia coli and Proteus when sowing the latter on a cup in the amount of 10/6 m.

Example 2

To prepare the environment SEDH-M used the above ingredients in the following concentrations, g / l: dry nutrient broth from pancreatic digest of Caspian sprat - 8.0; microbiological agar - 8.0; sucrose - 18.0; sodium chloride - 6.0; sodium carbonate - 0.7; sodium citrate - 8.0; sodium sulfite - 0.2; bromothymol blue - 0.7; mesoinositis - 0.7; EDTA iron (III) sodium salt - 0.4; activated carbon - 1.0; L-cysteine - 0.2; potassium tellurite - 0.001; bismuth pentahydrate nitrate - 0.06; cattle bile - 2.0.

Table 2 presents the characteristics of the growth and inhibitory environments SEDH-M, prepared according to the recipe of this example, and the environment of the prototype SEDH.

As can be seen from table 2, the environment SEDH-M of this composition in both growth and elective (inhibitory) properties corresponded to the required standards. Atypical colonies of cholera vibrios in the SEDH-M medium were not registered. Environment SEDH (prototype) in elective properties was ineffective.

Example 3

To prepare the environment SEDH-M used the above ingredients in the following concentrations, g / l: dry nutrient broth from pancreatic digest of Caspian sprat - 10.0; microbiological agar - 10.0; sucrose - 20.0; sodium chloride - 8.0; sodium carbonate - 0.8; sodium citrate - 10.0; sodium sulfite - 0.3; bromothymol blue - 0.8; mesoinositis - 0.8; EDTA iron (III) sodium salt - 0.5; activated carbon - 1.2; L-cysteine - 0.3; potassium tellurite - 0.002; bismuth pentahydrate nitrate - 0.08, cattle bile - 3.0. The technology for preparing the medium is the same as in example 1, 2.

Table 3 presents data on the growth and inhibitory properties of the SEDH-M media prepared according to the recipe of this example and the SEDH prototype medium.

As can be seen from table 3, the environment SEDH-M of this composition in both growth and elective properties corresponded to the required standards. Atypical colonies of cholera vibrios in the SEDH-M medium were not observed. The environment of SEDH by elective properties remained ineffective.

Example 4

To prepare the SEDH-M medium, the above-mentioned ingredients were used in the following concentrations, g / l: dry nutritional broth from pancreatic digest of Caspian sprat - 12.0; microbiological agar - 12.0; sucrose - 22.0; sodium chloride - 10.0; sodium carbonate - 0.9; sodium citrate - 12.0; sodium sulfite - 0.4; bromothymol blue - 1.0; mesoinositis - 1.0; EDTA iron (III) sodium salt - 0.6; activated carbon - 1.4; L-cysteine - 0.4; potassium tellurite - 0.0025; bismuth pentahydrate nitrate - 0.1; cattle bile - 4.0.

The medium was prepared as in examples 1, 2, 3. In a similar manner, the prototype SEDH medium was prepared in the original recipe. Sowing of two test cultures of cholera vibrio and two test cultures of commensal microflora and incubation of cups with crops was carried out according to the generally accepted methodology.

Table 4 shows the growth and inhibitory properties of the SEDH-M media prepared according to the recipe of the present example and the prototype SEDH medium. As can be seen from table 4, the environment SEDH-M of this composition in both growth and elective properties corresponded to the required standards. There were no atypical colonies of cholera vibrios on the SEDH-M medium. The environment of SEDH by elective properties remained ineffective.

Example 5

To prepare the environment SEDH-M used the above ingredients in the following maximum concentrations, g / l: dry nutrient broth from pancreatic digest of Caspian sprat - 14.0; microbiological agar - 14.0; sucrose - 24.0; sodium chloride - 13.0; sodium carbonate - 1.0; sodium citrate - 14.0; sodium sulfite - 0.5; bromothymol blue - 1.2; mesoinositis - 1.2; EDTA iron (III) sodium salt - 0.7; activated carbon - 1.6; L-cysteine - 0.5; potassium tellurite - 0.003; bismuth pentahydrate nitrate - 0.12; cattle bile - 5.0.

The technology for the preparation of the medium as in examples 1, 2, 3, 4. In a similar manner, the prototype medium SEDH was prepared in the original recipe. Sowing test cultures of cholera vibrio and test cultures of commensal microflora and incubating cups with crops were made according to the generally accepted methodology.

Table 5 presents the test results of the growth and inhibitory properties of the environment SEDH-M prepared according to the recipe of the present example, and the environment of the prototype SEDH. As can be seen from table 5, the environment SEDH-M of this composition in terms of growth and elective properties corresponded to the required standards. There were no atypical colonies of cholera vibrios on the SEDH-M medium. The environment of SEDH by elective properties remained ineffective.

Example 6

To prepare the SEDH-M medium, in this case, instead of a dry nutrient broth based on pancreatic digest of Caspian sprat, a dry nutrient broth was taken from a fish-bone meal hydrolyzate (see the Diagnostic Preparations product catalog, Obolensk, FBUN SSC PMB, 2012). while maintaining all the other ingredients in concentrations corresponding to example 3 (average concentration), g / l: dry nutrient broth from the hydrolyzate of fish-bone meal - 10.0; microbiological agar - 10.0; sucrose - 20.0; sodium chloride - 8.0; sodium carbonate - 0.8; sodium citrate - 10.0; sodium sulfite - 0.3; bromothymol blue - 0.8; mesoinositis - 0.8; EDTA iron (III) sodium salt - 0.5; activated carbon - 1.2; L-cysteine - 0.3; potassium tellurite - 0.002; bismuth pentahydrate nitrate - 0.08, cattle bile - 3.0.

These reagents were poured into 1.0 L of distilled water, dissolved with stirring and boiled for 20-30 minutes. The reaction medium was adjusted to 8.0. The prepared medium was poured into Petri dishes. Similarly, a prototype SEDH medium was prepared in the original recipe. Sowing of two test cultures of cholera vibrio and two test cultures of commensal microflora and incubation of cups with crops was carried out according to the generally accepted methodology.

Table 6 presents data on the growth and inhibitory properties of the SEDH-M medium.

As can be seen from table 6, the growth and elective properties of the SEDH-M medium were completely preserved and corresponded to the standards. The environment of SEDH by elective properties remained unsatisfactory. Thus, both nutrient bases provide good germination of cholera vibrio and sufficient inhibition of test microflora.

Example 7

In this case, the same recipe was used to prepare the SEDH-M medium as in Example 6, except that a combination of two components of the analog medium, the yeast extract and peptone, was used as a nutritional basis (see Microbiology Manual, Merek K GaA, Darmstadt, Deutschland, 1996 s. 250). The composition of the medium, g / l: yeast extract - 5.0; peptone - 10.0; microbiological agar - 10.0; sucrose - 20.0; sodium chloride - 8.0; sodium carbonate - 0.8; sodium citrate - 10.0; sodium sulfite - 0.3; bromothymol blue - 0.8; mesoinositis - 0.8; EDTA iron (III) sodium salt - 0.5; activated carbon - 1.2; L-cysteine - 0.3; potassium tellurite - 0.002; bismuth pentahydrate nitrate - 0.08; cattle bile - 3.0. The technology for preparing media and testing is the same as in the previous examples. The test results are presented in table 7.

As can be seen from table 7, the growth and elective properties of the SEDH-M medium in this case corresponded to modern requirements, while the SEDH medium in terms of its inhibitory properties remained unsatisfactory. The test results confirm the suitability of the yeast extract and peptone as a nutrient base for the desired environment SEDH-M.

Example 8

Growth properties of the SEDH-M medium, the SEDH prototype medium and the TCBS analogue medium in relation to seven field strains of cholera vibrios. As the main variant of the test medium, SEDH-M medium was taken, prepared according to the recipe of Example 3 using dry nutrient broth on Caspian sprat as a nutrient base. SEDH medium was prepared according to the original recipe; TCBS medium was prepared according to the manufacturer's recommendations (1). Vibrio cholerae cultures were grown for 24 hours on conventional alkaline agar (pH-7.6) and sown on agar plates of test media of 10 ² M.K. Crops were recorded after 1 day of incubation. The results of the experiment are presented in table 8. As can be seen from table 8, the growth of all seven field strains of cholera vibrios in all three environments was satisfactory.

Example 9

Experimental data on modeling the selection of cholera vibrioes from human feces are presented. Feces taken from a healthy person using TCBS were examined for the absence of vibrio flora, including cholera vibrios. Feces was artificially contaminated with all seven field strains of cholera vibrios with the expectation of obtaining a pathogen concentration of 10 ² m.k. in a volume of 0.2 ml of liquid, which amounted to the seed dose. Crops were performed on three media: SEDH-M, TCBS and SEDH. SEDH-M medium was prepared using dry nutrient broth on Caspian sprat digest, the concentration of medium components corresponded to the recipe of Example 3. SEDH and TCBS media were prepared as described in the previous example. SEDH and TCBS media with cultures according to the operating instructions for these media were incubated at 37 ° C, and SEDH-M medium was incubated at a temperature of 30 ° C, which is optimal for suppressing intestinal microflora.

Table 9 shows the results that demonstrate the effectiveness of the selection of cholera vibrios from artificially contaminated stool samples of a healthy person.

As can be seen from table 9, the environment SEDH-M and TCBS allowed to isolate the causative agent of cholera of all seven field strains when sowing very large quantities of native feces (0.2 ml). In all cases, cholera colonies without signs of growth of intestinal microflora grew on SEDH-M medium. Compared to SEDH-M, TCBS medium under these conditions was characterized by reduced growth and elective ability, which was expressed in fewer colonies of cholera vibrios growing on plates and the presence of a certain germination of intestinal microflora. The SEDH environment turned out to be absolutely non-elective. Thus, our proposed environment for the studied effect significantly exceeds the prototype environment and is more elective than the analog medium (TCBS).

Example 10

The data on modeling the selection of cholera vibrioes from water in a natural reservoir are presented. River water samples were taken in the spring and, using the TCBS medium, they were checked for the absence of vibrio microflora in it. Vibrio cholerae (seven field strains from the Rostov NIPCH collection) were introduced into water with the expectation of obtaining a pathogen concentration of 10 ² m.k. in a volume of 0.2 ml, constituting the seed dose. Crops were performed on three media: SEDH-M, TCBS and SEDH. Wednesday SEDH-M prepared according to the recipe of example 3 using a dry nutrient broth from a digest of Caspian sprat. Wednesday SADH and TCBS - according to the original recipe. Crops on SEDH and TCBS medium were incubated, as is customary, at 37 ° С for 1 day, on SEDH-M medium - incubated at 40 ° С, which in our experience is optimal for suppressing water microflora.

The results demonstrating the effect of the selection of cholera vibrios from contaminated water are presented in table 10.

As can be seen from table 10, both SEDH-M and TCBS media made it possible to isolate cholera vibrios from the studied samples, the latter being somewhat less effective. SEDH was weakly inhibitory and did not allow to achieve the desired result.

Example 11

The data on the field test of the SEDH-M medium are presented. A total of 80 samples of natural river water were examined (Don River), the survey period was May-August 2010. 0.5 ml of native river water was delivered to the laboratory, the plates were dried until the seeds were completely dried and incubated at 40 ° С for 1 day, after which they were viewed in a stereo microscope with oblique illumination. Red-orange colonies grown on SADH-M medium were agglutinated with O-1 cholera serum at a 1: 100 dilution. Colonies that gave positive agglutination with a negative control in a 0.9% NaCl solution were selected for further study and identification of cultures according to instructive documents according to the generally accepted scheme (5). As a result of examinations, 6 atoxigenic strains of V. cholerae were isolated, the properties of which are presented in Table 11. The cultures presented have typical vibrio properties and are classified as V. cholerae (atoxigenic, non-epidemic strains). Two strains show signs of a transition to a rough shape. Thus, direct evidence has been obtained of the suitability of the SEDH-M medium for the isolation of cholera vibrioes from environmental objects.

Thus, we can conclude that the results of complex tests of a new elective differential medium for the isolation of modified vibrio cholerae (SEDH-M) showed that the prototype environment of SEDH with the same growth qualities is significantly inferior to the desired medium in terms of inhibiting extraneous microflora activity. This found expression in the much greater efficiency of the SEDH-M medium in the isolation of cholera vibrioes from artificially contaminated samples of human feces and river water samples. The obtained results also showed that the SEDH-M medium was not inferior to the internationally known selective medium for the isolation of TCBS cholera vibrioes in model experiments with artificially contaminated samples of stool and water samples. SEDH-M medium can be produced in three versions with different nutritional bases: dry nutritional broth from pancreatic digest of Caspian sprats, dry nutritional broth from hydrolyzed fish-bone meal and yeast extract-peptone complex. SEDH-M medium can be used for the field isolation of cholera vibrioes both from human feces and from natural water during direct sowing of material and, naturally, when sowing after enrichment in peptone water.

The present invention was created due to a new composition of the medium, including active inhibitors of commensal intestinal and aqueous microflora, namely sodium citrate, bismuth nitrate, EDTA iron (III) sodium salt, cattle bile, potassium tellurite, bromothymol blue in high concentration with simultaneous stimulation growth of cholera vibrio under high pressure of the above inhibitors using mesoinositol, sodium sulfite, L-cysteine and activated carbon, which ultimately creates good growth and inhibitory the forces of the environment.

In this case, the medium creates a clear differentiating color effect: cholera vibrioes form red-orange colonies on a dark purple background, easily distinguishable by morphology and color from the few germinating colonies of other species. An important advantage of the SEDH-M medium is the interchangeability of its three different nutrient bases: dry nutrient broth from a Caspian sprat digest (NPO Nutrient Sredy, Makhachkala), dry nutrient broth from a fish-bone meal hydrolyzate (SSC PMB Obolensk) and the yeast extract-peptone complex produced by a number of foreign companies, which creates the possibility of economic maneuver in the manufacture of the medium.

Information sources

1. Standard Methods for the Examination of Water and Wastewater. 17 ed. Washington, D.C. American Public Health Association, 1989.

2. Temporary pharmacopoeial article of the VFS. 42-411 BC-93. Nutrient medium for the isolation of cholera vibrio Electron-differential dry (SEDH). Approved Deputy Minister of the Ministry of Health of the Russian Federation on July 19, 1993 Register of medicines. Means and products of honey. destination - No. 93/270/17.

3. Control of diagnostic nutrient media by biological indicators for the causative agents of plague, cholera, anthrax, tularemia, brucellosis, legionellosis. MU 3.3.2.2124-06, M., - 2006.

4. Methods for controlling bacteriological culture media. MUK 4.2.2316-08, M, - 2008.

5. Guidelines "Laboratory diagnosis of cholera": MU 4.2.2218-07, M., - 2007.

Table 1. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 0 ± 0.6 9 ± 1.2 V.cholerae O1 M-878 0 ± 0.8 16 ± 2 E.coli 18 P.vulgaris HX-19 0 0 120 380 Number 222 0 0 250 750 Prototype environment V.cholerae O1 P-1 0 24 ± 1 V.cholerae O1 M-878 5 ± 1 39 ± 4 E.coli 18 0 0 0 13 ± 1 P.vulgaris HX-19 0 20 ± 7 160 ± 30 > 200 Number 222

Table 2. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-l 3 ± 1 32 ± 3 V.cholerae O1 M-878 3 ± 1 38 ± 2 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-l 0 24 ± 1 V.cholerae O1 M-878 5 ± 1 39 ± 4 E.coli 18 0 0 0 33 ± 1 P.vulgaris HX-19 0 20 ± 7 160 ± 30 > 200 Number 222

Table 3. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 4 ± 1 35 ± 5 V.cholerae O1 M-878 3 ± 1 36 ± 3 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-1 2 ± 1 27 ± 2 V.cholerae O1 M-878 5 ± 1 30 ± 5 E.coli 18 0 0 40 ± 6 84 ± 10 P.vulgaris HX-19 0 78 ± 12 230 ± 30 > 300 Number 222

Table 4. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 2 ± 0.5 37 ± 5 V.cholerae O1 M-878 3 ± 0.6 31 ± 4 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-1 2 ± 0.5 19 ± 1 V.cholerae O1 M-878 2 ± 0.5 28 ± 6 E.coli 18 0 0 0 13 ± 1 P.vulgaris HX-19 0 34 ± 6 110 ± 28 160 ± 26 Number 222

Table 5. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 0 4 ± 1 V.cholerae O1 M-878 0 6 ± 1 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-1 2 ± 0.5 19 ± 1 V.cholerae O1 M-878 2 ± 0.5 28 ± 6 E.coli 18 0 0 0 13 ± 1 P.vulgaris HX-19 0 34 ± 6 110 ± 28 160 ± 26 Number 222

Table 6. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 5 ± 1 34 ± 5 V.cholerae O1 M-878 4 ± 1 35 ± 3 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-1 2 ± 1 27 ± 2 V.cholerae O1 M-878 5 ± 1 30 ± 5 E.coli 18 0 0 40 ± 6 84 ± 10 P.vulgaris HX-19 0 78 ± 12 230 ± 30 > 300 Number 222

Table 7. Test strain Growth properties of media - the number of colonies of cholera vibrios grown during sowing: Inhibitory properties of media - residual seedlings of commensal colonies during sowing: 10 m.k. 100 mk 10 ³ m.k. 10 ⁴ m.k. 10 ⁵ m.k. 10 ⁶ m.k. one 2 3 four 5 6 7 Wednesday SADH-M V.cholerae O1 P-1 6 ± 1 37 ± 5 V.cholerae O1 M-878 5 ± 1 38 ± 3 E.coli 18 0 0 0 0 P.vulgaris HX-19 0 0 0 0 Number 222 Prototype environment V.cholerae O1 P-1 2 ± 1 24 ± 2 V.cholerae O1 M-878 5 ± 1 31 ± 5 E.coli 18 0 0 40 ± 6 84 ± 10 P.vulgaris HX-19 0 78 ± 12 230 ± 30 > 300 Number 222

Table 8. No. p / p The name of the strain of cholera vibrios The number of colonies grown from 10 ² m.k. inoculum on media: SEDH-M SEDH TCBS one 2 3 four 5 one. V.cholerae O1 39 17 918 ctx- 42 ± 1 44 ± 1 30 ± 2 2. V.cholerae O1 39 16064 ctx + 39 ± 2 41 ± 3 34 ± 1 3. V.cholerae O1 26 ctx- 40 ± 2 42 ± 1 26 ± 3 four. V.cholerae O1 24 ctx- 35 ± 1 41 ± 2 27 ± 2 5. V.cholerae O1 12278 ctx + 40 ± 4 51 ± 1 26 ± 3 6. V.cholerae O1 43 ctx- 34 ± 1 45 ± 2 23 ± 2 7. V.cholerae non O1 16147 ctx- 45 ± 2 47 ± 1 24 ± 1

Table 9. No. p / p The name of the strain of cholera vibrios The growth of microflora on the media when sowing 0.2 ml of feces contaminated with cholera vibrios (10 ² m.k.) SEDH-M SEDH TCBS one 2 3 four 5 one. V.cholerae O1 39 17 918 ctx- 40 ± 1 colonies of cholera vibrios The growth of intestinal microflora 13 ± 2 colonies of cholera vibrios 2. V.cholerae O1 39 16064 ctx + 35 ± 1 colonies of cholera vibrios The growth of intestinal microflora Growth of intestinal microflora of 14 ± 1 colonies of cholera vibrios 3. V.cholerae O1 26 ctx- 30 ± 2 colonies of cholera vibrios The growth of intestinal microflora 16 ± 1 colonies of cholera vibrios four. V.cholerae O1 24 ctx- 35 ± 1 colonies of cholera vibrios The growth of intestinal microflora The growth of intestinal microflora 17 ± 2 colonies of cholera vibrios 5. V.cholerae O1 12278 ctx + 40 ± 1 colonies of cholera vibrios The growth of intestinal microflora 16 ± 32 colonies of cholera vibrios 6. V.cholerae O1 43 ctx- 24 ± 1 colonies of cholera vibrios The growth of intestinal microflora Growth of intestinal microflora 13 ± 1 colonies of cholera vibrios 7. V.cholerae non O1 16147 ctx- 35 ± 2 colonies of vibrios The growth of intestinal microflora The growth of intestinal microflora 14 ± 1 colonies of vibrios

Table 10. No. p / p The name of the strain of cholera vibrios The growth of microflora on the media when sowing 0.2 ml of river water contaminated with cholera vibrios (10 ² m.k.) SEDH-M SEDH TCBS one 2 3 four 5 one. V.cholerae O139 17918 ctx- 36 ± 1 colonies of cholera vibrios The growth of river microflora The growth of river microflora 14 ± 2 colonies of cholera vibrios 2. V.cholerae O139 16064 ctx + The growth of river microflora 30 ± 1 colonies of cholera vibrios The growth of river microflora The growth of river microflora 10 ± 1 colonies of cholera vibrios 3. V.cholerae O1 26 ctx- 30 ± 2 colonies of cholera vibrios The growth of river microflora The growth of river microflora 15 ± 1 colonies of cholera vibrios four. V.cholerae O1 24 ctx- The growth of river microflora 35 ± 1 colonies of cholera vibrios The growth of river microflora The growth of river microflora 19 ± 2 colonies of cholera vibrios 5. V.cholerae O1 12278 ctx + 41 ± 1 colonies of cholera vibrios The growth of river microflora 15 ± 2 colonies of cholera vibrios 6. V.cholerae O1 43 ctx- 25 ± 1 colonies of cholera vibrios The growth of river microflora The growth of river microflora 18 ± 1 colonies of cholera vibrios 7. V.cholerae non O1 16147 ctx- 25 ± 2 colonies of vibrios The growth of river microflora The growth of river microflora 19 ± 1 colonies of vibrios

Table 11. No. p / p Strain number Date of water sampling Crop properties Colony morphology Agglutability PCR Lum test with O1xc The presence of toxin (genotype) one 2 3 four 5 6 7 8 one 65-119-8 07/27/10 S O1 xc 1: 800 + + ctx- 2 66-125-3 07/27/10 S O1 xc 1: 400 + + 3 82-5-52 07/06/10 S O1 xc 1: 400 + + ctx- four 151-69-15 08/16/10 RS O1 xc 1: 800 RO xc 1: 100 + + ctx- 5 155-65-27 08/16/10 S O1 xc 1: 800 + + ctx- 6 155-65-29 07/16/10 RS O1 xc 1: 800 RO xc 1: 100 + + ctx-

Claims (3)

1. Elective differential nutrient medium for the isolation of cholera vibrios, containing a nutrient base, microbiological agar, sucrose, sodium chloride, sodium carbonate, potassium tellurite, bromothymol blue and distilled water, characterized in that nitrate is additionally used as inhibitors of extraneous microflora in the medium bismuth pentahydrate, sodium citrate, cattle bile, EDTA iron (III) - sodium salt, bromothymol blue are introduced in large quantities, and mesoino are introduced to stimulate the growth of cholera vibrio um, activated carbon, sodium sulfite and L-cysteine, wherein nutrient substrate is dry nutrient broth from pancreatic digest Caspian sprat the following component ratio, g / l:
pancreatic dry nutritional broth Caspian sprat 8.0-12.0 microbiological agar 8.0-12.0 sucrose 18.0-22.0 sodium chloride 6.0-10.0 sodium carbonate 0.7-0.9 sodium citrate 8.0-12.0 sodium sulfite 0.2-0.4 bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 activated carbon 1.0-1.4 L-cysteine 0.2-0.4 potassium tellurite 0.001-0.003 bismuth pentahydrate 0.06-0.1 cattle bile 2.0-4.0 distilled water 1 liter
at pH 8.0. 2. Elective differential nutrient medium for the isolation of cholera vibrios, containing a nutrient base, microbiological agar, sucrose, sodium chloride, sodium carbonate, potassium tellurite, bromothymol blue and distilled water, characterized in that nitrate is additionally used as inhibitors of extraneous microflora in the medium bismuth pentahydrate, sodium citrate, cattle bile, EDTA iron (III) - sodium salt, bromothymol blue are introduced in large quantities, and mesoino are introduced to stimulate the growth of cholera vibrio zit, activated carbon, sodium sulfite and L-cysteine, and as a nutritional basis, a dry nutrient broth from a fish-bone meal hydrolyzate was used in the following ratio of components, g / l:
hydrolyzed dry nutrient broth fish bone meal 8.0-12.0 microbiological agar 8.0-12.0 sucrose 18.0-22.0 sodium chloride 6.0-10.0 sodium carbonate 0.7-0.9 sodium citrate 8.0-12.0 sodium sulfite 0.2-0.4 bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 activated carbon 1.0-1.4 L-cysteine 0.2-0.4 potassium tellurite 0.001-0.003 bismuth pentahydrate 0.06-0.1 cattle bile 2.0-4.0 distilled water 1 liter
at pH 8.0. 3. Elective differential nutrient medium for the isolation of cholera vibrios, containing a nutrient base, microbiological agar, sucrose, sodium chloride, sodium carbonate, potassium tellurite, bromothymol blue and distilled water, characterized in that nitrate is additionally used as inhibitors of extraneous microflora in the medium bismuth pentahydrate, sodium citrate, cattle bile, EDTA iron (III) - sodium salt, bromothymol blue are introduced in large quantities, and mesoinosis is introduced to stimulate the growth of cholera vibrio um, activated carbon, sodium sulfite and L-cysteine, while the complex of yeast extract and peptone was used as a nutrient base in the following ratio of components, g / l:
yeast extract 5,0 peptone 10.0 microbiological agar 8.0-12.0 sucrose 18.0-22.0 sodium chloride 6.0-10.0 sodium carbonate 0.7-0.9 sodium citrate 8.0-12.0 sodium sulfite 0.2-0.4 bromothymol blue 0.6-1.0 Mesoinositis 0.6-1.0 EDTA iron (III) sodium salt 0.4-0.6 activated carbon 1.0-1.4 L-cysteine 0.2-0.4 potassium tellurite 0.001-0.003 bismuth pentahydrate 0.06-0.1 cattle bile 2.0-4.0 distilled water 1 liter
at pH 8.0.