RU2625869C1 - Method of sampling surface water reservoirs to determine presence of vibrio cholerae and portable device for its implementation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of water sampling from the surface of bodies of water to determine the presence of Vibrio cholerae comprises the descent device for the selection of the water using a rope to a depth of sampling. At the same time in the space of the reservoir taken with a portable device six samples using removable samplers, is a glass tube with a diameter of 5 mm and a length of 7 cm to form biofilms of V. cholerae. After 6 days lift device cable and tube are removed with forceps, placed into separate tubes containing 1% peptone water, capped and placed in a container for transportation to the laboratory for bacteriological testing. Portable apparatus for water sampling comprising a housing for containing the probe, which is a basis in the form of a disk configured with sockets diameter of 7 mm under the sampler in the form of glass tubes in an amount of six units, and a circular plate associated with the base vertical rod having a both sides of the thread at the end, with the nuts and cap fixed base and at the top by a washer and a nut plate on which is placed a float planted on the sleeve, and is fixed cap-holder with a hole for the cable. The base plate and the rigidity is further tightened by three screws.

EFFECT: increased efficiency of bacteriological research.

2 cl, 6 tbl, 3 ex

Description

The present invention relates to medical microbiology, and in particular to the field of obtaining and preparing sample samples from the water surfaces of reservoirs for bacteriological studies.

Currently, during the epidemic seasons, annually (May - October), depending on weather conditions, the problem of manually collecting water samples in natural reservoirs (seas, rivers, lakes, ponds), which is carried out once a week in liter containers (bathometers), is relevant. ) The latter are collected from all stationary points (at least 200 pieces) in a container to move pathogenic material and brought to the laboratory for subsequent biological examination of liquids for the presence of cholera vibrios. Selection technology is a routine, cumbersome and time-consuming process. Toxigenic vibrios are rarely released from the aquatic environment between cholera epidemics, but in recent years they have been found in accumulations of biofilms in sea and river water in Asia and Latin America (1, 2, 3, 4). The formation of biofilms by cholera vibrios is an important way of their survival in the environment; the latter can be located on the surface of algae, crustaceans, chitin and shields of crab, shrimp (5, 6).

In addition, biofilms provide a selective advantage to pathogenic vibrios, increasing their ability to spread under various environmental conditions. In this regard, it is important at the sampling stage to obtain a biofilm for its further research.

A known bathometer is a bottle on a rod of type GR-16M (see TU 52-05-62-64) containing a container in the form of a bottle inserted into a metal holder and fixed to it with a clamping tape and screw, while the holder with the bottle is installed and mounted on the rod, and the bottle is equipped with a metal head through which two tubes pass (water intake and air outlet).

The disadvantage of the bathometer is that with its help they take only one sample from one place, in which, as a rule, there is a low concentration of cholera vibrios and a large accompanying microflora that suppresses the growth of cholera vibrios.

Thus, the study of bathometers is inefficient when conducting continuous monitoring of water bodies for the presence of cholera vibrios.

Moreover, the isolation of single cells of cholera vibrios with minimal contamination of the sample is a laborious and lengthy process.

For the prototype, a method of sampling water was selected (see Pat. RU No. 2160891, class G01N 1/10, 12/20/2000), including lowering the bathometer using a cable and ballast weight to the depth of sampling, at which the sterile liquid is replaced by a sample followed by cooling to freezing temperature at a rate of at least 0.3 ° C per second.

In addition, the bathometer contains a housing on which the sampler is mounted with a lid that tightly closes the cavity of the sampler and a container with liquid nitrogen, the latter is connected via a valve to a heat exchanger located in the sampler between its frame and the cavity.

A water pump, a control unit connected with a sensor for immersion depth and controlling the valves of the water pump and the position of the cover of the sampler are located in the bathometer case.

The disadvantage of the prototype is its inefficiency in relation to sampling and research on the presence of cholera vibrios.

Freezing samples to 20-30 ° C negatively affects the viability of cholera vibrios, in accordance with MUK 4.2.2870-11 “Laboratory diagnosis of cholera,” a sample taken from water within 4 hours must be delivered to the laboratory for research. In addition, the device that implements the known method is difficult in design and unreliable in operation.

The technical task of the invention was to develop a method and device in the form of a portable structure for sampling from the water surface of water bodies, which allows to increase the efficiency of bacteriological studies for the presence of cholera vibrios.

The problem is achieved in that in the method of sampling water from the surface of reservoirs to determine the presence of cholera vibrios, including the descent of the device for sampling water using a cable to the depth of sampling, at the same time six samples are taken in a designated place of the reservoir using a portable device using interchangeable samplers, which are glass tubes with a diameter of 5 mm and a length of 7 cm for the formation of biofilms of cholera vibrios, then for 6 days, depending on the temperature changes in the environment conductive medium, a cable assembly is raised and the tube is removed with forceps, placed into separate tubes containing 1% peptone water, capped and placed in a container for transportation to the laboratory for bacteriological examinations, and to place the extracted set new samplers.

The proposed method can be implemented using a portable device for sampling water, consisting of a housing for placing a sampler in it, the housing being a base in the form of a disk made with nests with a diameter of 7 mm for samplers in the form of glass tubes in the amount of six pieces, and a round plate connected to the base with a vertical rod having a thread on both sides, on which the base is fixed below with nuts and cap, and the plate above which zmeschen float planted on the sleeve, and a cap-holder is fixed with a hole for the cable, wherein the base plate and the rigidity is further tightened by three screws.

The essence of the invention is illustrated by drawings, where

FIG. 1 shows a general view of a sampling apparatus;

FIG. 2 - view A;

FIG. 3 - section BB;

FIG. 4 - section bb;

FIG. 5 - section GG.

The device is a base 1, made in the form of a disk with a side 2 and six sockets 3, with a diameter of 7 mm and a depth of 10 mm for samplers 4 (see Fig. 1, 3, 4). The latter are glass tubes 7 cm long and 5 mm in diameter.

Using the rod 5 mounted vertically, the base 1 is connected to a round plate 6 having through holes in the amount of 6 pieces and fixed at a distance of H = 5.5 cm from the base 1 (see Fig. 2).

The rod 5 is made on both sides with a thread, on which the base 1 is supported by the washer 1 on the bottom with the washer 7, nuts 8, 9 (see Fig. 1) of the cap 1. On the opposite side of the rod 5, the plate 6 is fixed at the top through the nut 11, washer 12 A float 13 is placed above the latter, mounted on a sleeve 14 (see Fig. 5) and made of porous material (cork, sponge, wood and any materials with a specific gravity less than water) (see Fig. 1). The float 13 is fixed by a cap-holder 15 with a hole for the cable 16.

In this case, the plate 6 and the base 1 for structural rigidity are tightened with three screws 17 and secured with nuts 18 (see Fig. 4).

The operation of the device is as follows.

A device with 6 samplers placed in it 4 in the amount of 6 pieces is lowered using a cable 16 into a reservoir to a depth sufficient for sampling. Thanks to the float 13, the device under its weight does not sink to the bottom, but is held in the upper layer of the water surface, where the highest water temperature is observed. In addition, the cable allows you to adjust the required depth from the surface of the reservoir and fix the device in a stationary position. Then the device fixed by means of a cable 16 is left for several days at least 6 days in permanently allocated places depending on the temperature changes in the environment.

Thus, sampling is carried out at least once a week, to do this, raise the cable 16, and with the help of tweezers collect the samplers 4, placing each of them in a separate tube with 1% peptone water, and close the lid.

Then, new samplers are installed in place of the samplers taken for research, so that they simultaneously pass through the through holes of the plate 6 and are fixed in the nests 3 of the base 1.

After that, the tubes with samplers are transported to the laboratory for bacteriological studies.

As a rule, a biofilm is already formed on the surface of glass tubes during the period of being in water, which is directly studied in the laboratory for the presence of both cholera vibrioes and other pathogenic microorganisms (V. parahaemoliticus, E. coli, Enterococcus spp., Etc. .).

Due to the adhesion of microorganisms on the walls of the sampler and the formation of a biofilm on its surface, it becomes possible to study both the qualitative and quantitative composition of the microbiota of the reservoir, as well as the phage composition.

Example 1

In June 2015, at the stationary point of the river. Don a prototype sampler was installed; the exposure time in water was 24 hours. After extraction from the river, the samples were placed in an appropriate nutrient medium for further microbiological studies. At the same time, a water sample was taken from the same point and investigated according to the classical scheme recommended by MUK 4.2.2870-11.

The results of testing a new selection method and device for its implementation showed (see tables 1, 2) that during bacteriological examination, the total number of microorganisms in CFU accumulated in the samplers significantly exceeded CFU in water samples taken in a known manner. The trend continued with respect to cholera vibrios. If microorganisms of the Vibrio genus accumulated up to 50-70% of the total number of oxide-positive microflora in the samplers, then in the samples from water it was 20-35%, respectively.

Example 2

In July 2015, at the stationary point of the river. Temernik installed a prototype sampler; the exposure time in water was 24 hours. After extraction from the river, the sample was placed in an appropriate nutrient medium for further microbiological studies. At the same time, a water sample was taken from the same point and investigated according to the classical scheme recommended by MUK 4.2.2870-11.

The results of the studies (see tables 3, 4)

From tables 3, 4 it is seen that, along with the identification of vibrios belonging to non-O1 / non O139 serogroups, a cholera vibrio, assigned to the O1 serogroup, Serovar Inaba, was detected in a water sample selected in a known manner. A similar microorganism was isolated from the sampler.

Example 3

In August 2015, at the stationary point of the river. Temernik in the area of the Botanical Garden, a prototype sampler was installed, the exposure time in water was 24 hours. After extraction from the river, the sample was placed in an appropriate nutrient medium for further microbiological studies. At the same time, a water sample was taken from the same point and investigated according to the classical scheme recommended by MUK 4.2.2870-11.

From the results of the studies (see tables 5, 6) it can be seen that, thanks to the new method of water withdrawal, a virulent strain of V. cholerae O1 serogroup, Serovar Inaba, was found in the biofilm already in a day. It was not possible to isolate this cholera vibrio in a water sample taken in a known manner. This fact indicates that the more isolates (samples) are studied, the more likely it is to track the vibrio landscape of a surface reservoir at a given stationary point.

The use of the present invention allows, due to the portable design of the device for sampling water, to implement a new sampling technology with the creation of biofilms of cholera vibrios in vivo, since it is easier and faster to determine their viability in the composition of biofilms, which increases the efficiency of bacteriological studies for the presence of cholera vibrios.

In addition, the simultaneous taking of several samples makes it possible to conduct studies for the presence of other pathogenic organisms in the water, namely E. coli, enterococci.

Information sources

1. Alam M., Sultana M., Nair G. B. et al. // Appl. Environ. Microbiol. - 2006. - Vol. 72, No. 4. - P. 2849-2855.

2. Binsztein N., Costagligla, Pitchel M. // Appl. Environ. Microbiol. - 2004. - Vol. 70, No. 12. - P. 7481-7486.

3. Davey M.E., O’Toole G.A. // Microbiol. Mol. Biol. Rev. - 2000. - Vol. 64, - P. 847-867.

4. Hall-Stoodley Z., Stoodley P. // Trends Microbiol. - 2005. - Vol. 13, - P. No. 7-10.

5. Harrison-Balestra C., A.L. Cazzaniga, S.C. Davis, et al. // Dermatol. Surg. - 2003 - Vol. 29, p. 631-635.

6. Karatan E., Duncan T.R., Watnick P.I. // J. Bacteriol. - 2005. - Vol. 187, No. 21. - P. 7434-7443.

Claims (2)

1. The method of sampling water from the surface of reservoirs to determine the presence of cholera vibrios, including the descent of a device for sampling water using a cable to the depth of sampling, characterized in that at the same time in a designated area of the reservoir, six samples are taken using a portable device using interchangeable samplers representing glass tubes with a diameter of 5 mm and a length of 7 cm for the formation of biofilms of cholera vibrios, then after 6 days, depending on the temperature changes in the environment, the device the wasps are lifted and the tubes are removed with tweezers, placed in separate tubes with 1% peptone water, closed with a lid and installed in a container for transportation to the laboratory for bacteriological studies, and new ones are installed in the place of the extracted samplers. 2. A portable device for sampling water, consisting of a housing for placing a sampler in it, characterized in that the housing of the device is a base in the form of a disk made with nests with a diameter of 7 mm for samplers in the form of glass tubes in the amount of six pieces, and round a plate connected to the base with a vertical rod having a thread on both sides, on which the base is fixed below with nuts and a cap, and at the top by means of a washer and nut, the plate over which the float is placed th onto the sleeve, and is fixed with a cap-holder with a hole for the cable, while the base and plate for rigidity are additionally tightened with three screws.

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