RU82859U1 - DEVICE FOR BIOTESTING OF TOXICITY OR BIOLOGICAL ACTIVITY OF THE SOLUTION IN STUDY - Google Patents

Abstract

1. A device for biotesting the toxicity or biological activity of the test solution using planarians, including a stage, an optical microscope with an integrated video camera, a signal conversion unit, a computer, as well as a decapitation unit containing an optical element and a decapitation cuvette, made in the form of a container with a solution for placing a planarium, characterized in that the decapitation unit further comprises a control unit and a decapitation unit, wherein the decapitation cuvette is located on the upper surface o The new unit is a decapitation unit, to the bottom of which a cooled unit is attached, in the base case there is a light source that is configured to illuminate the planar receiving area, and a cooled unit that is vertically movable between the working surface of the cooling unit and the rear surface of the cell in the decapitation area and cooling the decapitation zone of the planarium, the inputs of the light source and the cooling unit being connected to the outputs of the control unit, the inputs of which are connected to the control panel and / or to a computer, where the control unit is additionally equipped with a communication interface. ! 2. The device according to claim 1, characterized in that the decapitation cuvette is made in the form of a planar element, and the total liquid volume is divided into three volumes sequentially connected to each other with a depth of 0.5 to 3.0 mm, located along the central axis of the decapitation cuvette made in the form of a reception zone, an orientation zone and a decapitation zone, moreover, the liquid output of the reception zone is connected to the liquid input of the orientation zone, the liquid output of which is connected to the liquid

Description

A device for biotesting toxicity or biological activity of a test solution.

Field of Invention

A utility model relates to a biotesting device. In particular, the device can be used to determine the biological activity or toxicity of the components that make up water, drugs, food or biological active additives (BAA) to food. The device contains a complex of hardware and software based on a computer, a microscope and additional nodes that increase the reproducibility of the results during mass screening of the investigated substances.

State of the art

Known methods of biotesting in which flatworms are used as test organisms - planaria [1, 2, 3].

To determine the impact of the environment on aquatic organisms, parameters are measured that characterize: survival, behavioral characteristics, morphological characteristics. One of the morphological parameters is the rate of regeneration of hydrobionts.

Known guidelines for the experimental determination of the genetic hazard and toxicity of waste entering the environment [4], based on determining the survival rate of aquatic organisms (planaria) and determining the rate of regeneration after decapitation of animals (separation of the head of the body), in which, as devices for decapitation, suggested to use petri dishes.

A device is known by which the behavioral characteristics of test organisms are studied [5]. Using the device, preliminary training of test organisms is carried out, applying light and current stimuli to them. The device allows you to measure the speed of movement of the planariums and compares the speeds of movement of different groups of regenerating planarians, for example, regenerating in a clean environment and in an environment containing the test substance. However behavioral measurement techniques

reactions, in general, have low accuracy and are able to register only high concentrations of the studied solutions.

Known more advanced devices and diagnostic methods associated with the registration of the dynamics of regeneration of planaria and based on the method of intravital morphometry of regeneration of planaria [6]. Intravital morphometry is based on recording photocontrast between the old (i.e., pigmented) and the new transparent part of the body (blastema) of the planaria.

The regenerated part of the body of the planaria - blastema is measured during the regeneration process either by directly measuring the linear dimensions of the planaria using an eyepiece micrometer [4], or the sizes of the planaria blastema are determined by their photographs, or the process of measuring the sizes and / or areas of the blastema and planaria is carried out using computer methods. Intravital research allows you to work with the same group of animals during the entire regeneration process, which improves the quality of measurement.

A device is known for a computer method for measuring the morphological parameters of a planarium body using a digitizer [2]. A more effective device for measuring the morphological parameters of a planarium body containing a video camera connected to a computer [7, 8]. The experimental setup contains a VAT 127-LH video camera mounted on an MBS-2 binocular microscope and connected to an IBM PC 486 AT computer [7].

One of the drawbacks of the considered devices, including computer morphometry devices, is that decapitation is also carried out in Petri dishes, which does not allow standardizing the planar decapitation procedure and increasing the reproducibility of the decapitation process.

It is known [9] that decapitation is carried out under a binocular microscope using an eye scalpel. The planarium transection operation is carried out in different areas of the planarium body, cutting the body in front of or behind the pharyngeal planarium [10]. The guidelines suggest decapitation at eye level [4].

Known devices for decapitation are made on the basis of a Petri dish filled with a layer of water in which the planarium is placed. Planaria move freely along the bottom of the Petri dish and change their body position. During decapitation, the operator selects an individual planarium and orient its position in the horizontal plane by rotating the Petri dish with planarians placed in it. In this case, the operator finds a position of the Petri dish in which the selected individual planarium occupies a convenient position for decapitation.

Planaria do not have a rigid skeleton and are mobile in solution, therefore, to limit their activity, before decapitation, they lower the ambient temperature (solution) in a Petri dish in which planaria are located by immersing the plate with the planaria in ice solution. The cooling of the planaria occurs over a sufficiently long period of time. During this time, planarians, in the initial state, in an expanded and flat form, manage to shrink and group their body, which greatly complicates the determination of the place where decapitation should be carried out. This, in turn, leads to large errors in determining the location of decapitation. Planaria with different decapitation positions subsequently regenerate at different speeds, which, in turn, leads to errors in determining the regeneration parameters and, consequently, to errors in diagnosis.

A device for decapitation [9], containing a paper filter placed in a Petri dish. A planarium is preliminarily placed on the surface of the filter. Before decapitation, the device is cooled on ice to partially immobilize planarians. However, applying planariums to the filter does not guarantee that planarians will not shrink when exposed to low temperature.

It is known that for a reliable experiment and reproducible results it is necessary to use a group of planaria in the amount of 25-30 individuals. Moreover, like all studies on hydrobionts, experiments must be carried out in 3-6 replicates [10]. Decapitation of such an amount of planaria is a rather laborious and lengthy procedure.

The technical problem is to increase the efficiency of the device for biotesting and rapid assessment of chemical and biologically active solutions in mass studies.

The next technical task is to create a decapitation unit, which provides higher reproducibility of the process of decapitation of hydrobionts.

Another technical task is to create a device containing a new decapitation unit, in which the process of decapitation of hydrobionts takes place in conditions that ensure self-orientation of the planaria in a position convenient for decapitation.

The essence of the technical solution

The technical problem is solved by the fact that a device has been developed for biotesting the toxicity or biological activity of the test solution using planaria. The device includes a stage, an optical microscope with an integrated video camera, a signal conversion unit, a computer, as well as a decapitation unit containing an optical element and a decapitation cuvette, made in the form of a flat container with a solution for placing planariums, the decapitation unit additionally containing a decapitation unit and a control unit . The decapitation unit, in addition to the decapitation cuvette, additionally contains a base, a cooled element, a cooling unit. The decapitation cuvette is placed on the surface of the base, in the body of which a light source and a cooled element are located, which is arranged to vertically move between the working surface of the cooling unit and the rear surface of the decapitation cuvette. The inputs of the light source and the cooling unit are connected to the outputs of the control unit, the inputs of which are connected to the control panel and / or to the system computer, where the control unit is additionally equipped with a power source.

Another aspect of the utility model is that the decapitation cuvette is made in the form of a planar element, and the total liquid volume is divided into three successively connected volumes from 0.5 to 3.0 mm deep, located along the central axis of the decapitation unit, made in the form reception zones, orientation zones and decapitation zones, where the orientation zone is made in the form of a channel with a width of 3 to 5 mm. Moreover, the liquid output of the reception zone is connected to the liquid input of the orientation zone, the liquid output of which is connected to the liquid input of the decapitation zone, where the reception zone is made with the possibility of lighting or darkening, and the lower surface of the decapitation cell placed under the decapitation zone is made with the possibility of cooling due to the cooled item.

Another aspect of the utility model is that the decapitation cuvette further comprises a top cover with the possibility of horizontal movement above the working surface of the decapitation unit and opening the receiving zone or decapitation zone with the possibility of input and / or output of solutions and planaria. Moreover, the reception zone is made in the form of flat liquid volumes, the cross section of which represents a circle, a rectangle, a square, a triangle, a polygon, an ellipse.

Another aspect of the utility model is related to the fact that the optical axis of the light source located at the base of the decapitation unit is aligned with the receiving zone of the planaria. The light source forms a luminous flux in the receiving zone of the planarium in the range from 300 to 800 nm and an intensity of from 200 to 2000 lumens.

Another aspect of the utility model is that for cooling the decapitation zone, the decapitation unit is provided with a clamping lever for moving the cooled element between the rear surface of the decapitation cuvette and the working surface of the cooling unit. The cooling unit is made on the basis of a Peltier element or contains channels for air or liquid cooling flow with the possibility of lowering the temperature of the cooled element to a value from -4 to + 8 ° C.

Another aspect of the device is that the material of the decapitation cuvette is selected from glass and polymer. Moreover, the polymers are selected from the group consisting of polycarbonate, polymethylmethacrylate, while the thickness of the decapitation cuvette is from 1.0 to 5 mm.

List of figures

Figure 1. Block diagram of a device for biotesting.

Figure 2. Section decapitation. A light source illuminates the cuvette from below.

Figure 3. Top view of decapitation unit.

Figure 4. Appearance of the decapitation cuvette

Utility Model Description

Figure 1 shows a block diagram of a device for biotesting toxicity or biological activity of a test solution using planaria, using a computer method for measuring the morphological parameters of the body of planaria. The device includes a container 11 for growing planarium 1, glasses 12 with test solutions, into which decapitated planarium 2 is placed, a stage 16 on which a Petri dish 42 with regenerated planarium 3 is placed, a microscope combined with a video camera 17, signal matching unit 18, a computer 19, a display 20 and a decapitation unit 10, which includes a decapitation unit 13, an optical element 14, a control unit 15. The optical element 14 can be made on the basis of a binocular microscope. The control unit 15 contains an interface 28 containing an input for connecting a decapitation unit 10 to a computer 19, and is also connected to an input of a built-in or remote keyboard

27 to control the inclusion of the light source 23 and to control the cooling unit 25 included in the decapitation unit 13.

Figure 2 shows a sectional view of the decapitation unit 13, which is part of the decapitation unit 10. The decapitation unit 13 contains: a base 21, a decapitation cuvette 22, a light source 23, a cooled element 24, a cooling unit 25, a radiator 26.

Figure 3 shows a top view of the decapitation unit 13, which shows the location of the decapitation cuvette 22, which is placed on the working surface 29 of the base 21 and pressed to the base using clamps 30. The stops 31-34 serve to limit the movement of the cover 35 in the horizontal direction ( see figure 2).

Figure 4 shows a view of the working surface 36 of the decapitation cell. The design of the decapitation cuvette 22, in which the planarium is decapitated, contains three fluid zones sequentially connected to each other: the receiving zone 37, in which the planarium 1 intended for decapitation is placed, the orientation zone of the position of the planarium 38 and the decapitation zone 39. Moreover, the liquid output of the receiving zone 37 is connected to the liquid inlet of the orientation zone 38, the output of which is connected to the inlet of the decapitation zone 39, thus three volumes sequentially connected to each other with a depth of 0.5 to 3.0 mm located along the central axis of the decapitation cell. In this case, the receiving zone is made with the possibility of lighting or darkening, and the lower surface of the decapitation cell placed under the decapitation zone is made with the possibility of cooling due to the cooled element. The decapitation cuvette is made with the possibility of input and / or output of solutions and input and / or output of planaria. To this end, the decapitation cell further comprises a top cover with the possibility of its horizontal movement above the working surface of the decapitation cell and the possibility of opening the receiving zone or decapitation zone.

A device for biotesting toxicity or biological activity of a test solution using planaria works as follows. Planaria 1 contain in containers 11 with a volume of 3-5 liters at a temperature of culture water from 19 to 20 ° C. Cultural water is a mixture of distilled and tap water in a ratio of 2: 1. The planarium environment may be water exposed to physical factors, including

the number of magnetic fields, electromagnetic fields and radiation, light radiation, sound effects, radiation, or combinations thereof.

For diagnosis, planaria are used after 5-8 days of fasting, more preferably after 7 days of fasting. Planaria 1 is removed from the container 11 and placed in the reception zone 37 of the decapitation cuvette 22. Using the lid 35, the reception zone 37 and the orientation zone 38 are closed from the influence of external lighting. The planaria calms down, after which the operator turns on the light source 23. In this case, the light exposure in the reception zone is carried out by a light flux in the range from 300 to 800 nm and an intensity from 200 to 2000 lumens.

The planaria leaves the illuminated section of the decapitation cell zone 37 and through the orientation zone 38 enters the decapitation zone 39. The operator cools the decapitation zone 39 using a cooled element 24, which is pressed against the rear surface of the decapitation cell 41 in the decapitation zone using the lever 40 to move the cooled element between the back surface of the decapitation cell and the working surface of the cooling unit. The cooling unit is made on the basis of a Peltier element or contains channels for air or liquid cooling flow with the possibility of lowering the temperature of the cooled element to a value from -4 to + 8 ° C.

The decrease in temperature of the solution in the decapitation zone 39 relative to the environment occurs to a value of from +4 to + 15 ° C. From the action of the lowered temperature, the planaria freezes, and the operator, observing through the optical element of the microscope 14, accurately determines the place of decapitation, which most optimally passes along the trajectory of the planar's eyes. After decapitation is completed, the cooled element 24 by means of the lever 40 is returned to its original state and pressed against the working surface of the cooling unit 25.

The decapitated planarium 2 is placed in glasses 12 with the studied liquids, in which the process of their regeneration takes place. As the studied solutions can be used solutions containing chemical or biologically active substances that are part of drugs, biologically active food additives, veterinary drugs, homeopathic remedies. As the studied solutions, extracts from food products or additives to them, soil samples, as well as extracts from polymeric materials, cosmetics, atmospheric pollution, extracts from plant materials can be used. As the test solution, different types of water can be used, including bottled water, as well as

solutions of environmental pollutants and their mixtures, including industrial and municipal effluents containing oil products and heavy metals, which allows monitoring of the ecological state of the environment.

Due to cell proliferation during the regeneration of planaria, blastema grows. To determine the regeneration parameters, the planarium regenerating 3 are placed in Petri dishes 42, placed on the stage 16 of the microscope 17, equipped with a video camera. The analog output of the video camera is connected to the input of the matching node 18, the output of which is connected to the computer 19. Instead of an analog camera, you can use a digital video camera using the digital input of the computer. The resulting video image of the regenerating planarium is stored and transmitted to the display 20 and to the database. Using the image on the display, the operator selects the most suitable frame and saves data about a particular planarium by assigning it a number. Using software, the operator determines the size of the area of the old (residual) and regenerated (new) part of the planarium body and calculates the indicator “regeneration criterion”, which is the ratio of the blastema area to the total area of the planaria. The regeneration criterion (expressed as a percentage) is calculated by the formula: R = s / S, where s is the area of the blastema, S is the area of the entire body of the regenerated planaria at a given time.

Biotesting of chemical and / or biologically active substances is mainly carried out using hydrobionts included in the group of planaria Dugesia (Girardia) tigrina, Schmidtea mediterranea. The analysis data are compared with the regeneration data of planaria, which are placed in a glass of clean water. The results of biotesting are obtained by analyzing the data of calculation of the indicator “criterion of regeneration” from 20 to 30 individuals of planaria. Next, determine the average value of the regeneration criterion in the control and diagnosed groups and draw conclusions about the toxicity or biological activity (regenerative ability) of the test solution.

Data on the results of studies are entered into computer 19, which allows creating problem-oriented data banks that can be used in the development and testing of new drugs, biologically active food supplements, as well as creating databases for predicting possible environmental situations.

Presented in figure 4, the placement and shape of the zones in the decapitation cell includes, but is not limited to other options. For example, the receiving zone 37 may be made in the form of a circle, rectangle, square, triangle, polygon or ellipse. The width of the decapitation zone 38 may be equal to or greater than the width of the orientation zone 39.

The material of the decapitation cuvette 22 is selected from glass or polymer. The thickness of the decapitation cell is from 1.0 to 5 mm. Polymers are preferably used as the material for the decapitation cuvette.

The choice of the material of the decapitation cuvette is based on the possibility of stamping or the formation in the molten state of the volume of liquid zones on the surface of the decapitation cuvette, as well as on the basis of the ability of the decapitation cuvette material to withstand temperature cycles. Thermoplastic plastics provide the ability to stamp and form the selected shape of the planar element from the polymer melt. The polymers are included in the group consisting of polycarbonate, polymethyl methacrylate. Polymethyl methacrylate and a transparent cuvette material are preferred.

The decapitation cuvette 22 is installed on the working surface 29 of the base 21, equipped with protrusions 32, 34, limiting the position of the decapitation cuvette 22. The decapitation cuvette 22 is pressed against the surface of the base 29 using clamps 30, the appearance of which is shown in FIG. 3. When installing the decapitation cuvette 22, the optical axis 43 of the light source 23 is located in the receiving zone 37, and the axis 44 of the cooled element 24 is aligned with the decapitation zone 39.

A feature of the decapitation unit under consideration is that during the decapitation process, not the entire decapitation cell 22 with the planarium located in it is cooled, but only an insignificant part of the decapitation cell located in decapitation zone 39. Moreover, for the immobilization of planarium 1, not a prolonged exposure to low temperature was used, but a pulsed low-temperature effect of the cooled element 24 on the decapitation zone 39 of the decapitation cuvette 22.

When the light source 23 is turned on and the zone 37 is illuminated, the planarium, avoiding lighting, moves to the orientation zone 38 and moves head first along the zone. Orientation of the planaria parallel to the axis 45 of the decapitation cuvette 22 improves the accuracy and reproducibility of the decapitation operation.

Example 1. Checking the scatter of experimental data during decapitation of planaria at eye level compared with the result of decapitation on other parts of the head end of the body of planaria

The planaria is placed in the decapitation device described above and an operation is performed to remove the front end of the planaria body. The decapitation operation is carried out under the control of a binocular microscope with an eye scalpel in non-sterile conditions. The operation is performed at the moment of creeping of the planaria in the field of view of the binocular microscope, when it freezes from the rapid cooling of the water after switching on the Peltier element.

The advantage of this device is the stable stop of the planarians in the field of view of the microscope, which allows you to make an accurate cut and ensure the reproducibility of the procedure for separating the head from the body of the planarians. The operation is carried out in a standard place: at the eye level of the planaria.

The importance of ensuring the reproducibility and accuracy of the decapitation operation is reflected in the experimental results presented in table 1.

As can be seen from table 1, when performing the operation in the “standard” (by eye level) and “non-standard place”, the values of the regeneration criterion in a statistically significant way (P <0.02) differed from each other.

Due to the accuracy of the operation, the spread of experimental data is also reduced. So, the standard error of the arithmetic mean value (m) for the indicator “regeneration criterion” decreased from 0.065 (decapitation in a “non-standard place”, to 0.039 (decapitation at eye level).

Thus, these results emphasize the importance of reproducibility and accuracy of the decapitation operation, which provides the design of the decapitation unit.

Table 1. Morphometric indicators of planarium regeneration after decapitation according to eye level or when decapitating in a “non-standard place” Morphometric indicators Statistical indicators Eye level decapitation (n = 24) Decapitation in a “non-standard place” (n = 27) Area of decapitated planarium, conventional units M ± m 8212.0 ± 276.5 8173.6 ± 365.8 t 0.08 R Blastema area M ± m 99.90 ± 4.2 114.9 ± 7.3 t 1.72

conv.ed R The criterion of regeneration,% M ± t 1.22 ± 0.039 1.41 ± 0.065 t 2,39 R <0.02

n is the number of animals in the group;

M is the arithmetic mean value;

m is the standard error of the arithmetic mean;

t is the value of the Fisher-Student t criterion;

P is an indicator of the level of significance of differences between control and experience.

Industrial applicability

The device is convenient in operation and provides effective and reproducible testing of test solutions containing chemical or biologically active substances. The device provides the possibility of self-orientation of the planaria, their exit to the decapitation zone in a position convenient for decapitation, and the possibility of quick immobilization of the planaria by cooling the decapitation zone.

Literature

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2. Tiras H.P., Khachko V.I. Criteria and methods for the regeneration of planaria. //Ontogenesis. - 1990. - T. 21, No. 6. - S.620-624.

3. Proynova V.A., Tiras H.P., Rozhnov G.I. Biotesting on Dugesia tigrina planarians - opportunities and prospects for environmental hygiene. // In the book: 2nd Congress of Russian Toxicologists. (November 10-13, 2003). Abstracts of reports. - M. - Russian Register of Potentially Hazardous Chemical and Biological Substances. - 2003. - S.211-213.

4. The methodological basis of biotesting and determination of the genetic hazard of waste entering the environment. Guidelines RD-64-085-89 (06/01/1990).

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6. Tiras H.P., Sakharova N.Yu. Intravital morphometry of planarium regeneration. //Ontogenesis. - 1984. - T. 15, No. 1. - S. 41-48.

7. Karagodin VP, Chertkoyeva ZA. The possibility of using biotesting for a preliminary assessment of the effectiveness of dietary supplements. (http://www.sds-bio.org/bb/bb10-06.doc) (2006).

8. Ermakov A.M. et al. Modulation of the proliferative activity of stem cells of regenerating planaria using magnetic fields and pharmacological agents. Abstracts of the report. Moscow (http://www.cmbt.su/rus/science/science252.html) (2007.).

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Claims (15)

1. A device for biotesting the toxicity or biological activity of the test solution using planarians, including a stage, an optical microscope with an integrated video camera, a signal conversion unit, a computer, as well as a decapitation unit containing an optical element and a decapitation cuvette, made in the form of a container with a solution for placing a planarium, characterized in that the decapitation unit further comprises a control unit and a decapitation unit, wherein the decapitation cuvette is located on the upper surface o The new unit is a decapitation unit, to the bottom of which a cooled unit is attached, in the base case there is a light source that is configured to illuminate the planar receiving area, and a cooled unit that is vertically movable between the working surface of the cooling unit and the rear surface of the cell in the decapitation area and cooling the decapitation zone of the planarium, the inputs of the light source and the cooling unit being connected to the outputs of the control unit, the inputs of which are connected to the control panel and / or to a computer, where the control unit is additionally equipped with a communication interface. 2. The device according to claim 1, characterized in that the decapitation cuvette is made in the form of a planar element, and the total liquid volume is divided into three volumes sequentially connected to each other with a depth of 0.5 to 3.0 mm, located along the central axis of the decapitation cuvette made in the form of a reception zone, an orientation zone and a decapitation zone, moreover, the liquid output of the reception zone is connected to the liquid input of the orientation zone, the liquid output of which is connected to the liquid input of the decapitation zone, where the reception zone is made with the possibility of lighting I or dimming, and the lower surface of the decapitation cell, placed under the decapitation zone, is made with the possibility of cooling due to the cooled element. 3. The device according to claim 2, characterized in that the decapitation cell is made with the possibility of input and / or output of solutions and planaria. 4. The device according to claim 3, characterized in that the decapitation cell further comprises a top cover with the possibility of its horizontal movement above the working surface of the decapitation cell and the possibility of opening the receiving zone or decapitation zone. 5. The device according to claim 2, characterized in that the reception zone is made in the form of flat liquid volumes, the cross section of which represents a circle, rectangle, square, triangle, polygon, ellipse. 6. The device according to claim 3, characterized in that the orientation zone is made in the form of a channel with a width of 3 to 5 mm. 7. The device according to claim 2, characterized in that the thickness of the decapitation cell is from 1.0 to 5 mm. 8. The device according to claim 1, characterized in that the optical axis of the light source located at the base of the decapitation unit is aligned with the receiving zone of the planaria. 9. The device according to claim 8, characterized in that the light source forms a luminous flux in the receiving zone of the planarium in the range from 300 to 800 nm and an intensity of from 200 to 2000 lumens. 10. The device according to claim 1, characterized in that for the possibility of cooling the decapitation zone, the decapitation unit is equipped with a clamping lever for moving the cooled element between the rear surface of the decapitation cuvette and the working surface of the cooling unit. 11. The device according to claim 10, characterized in that the cooling unit is based on a Peltier element or contains channels for air or liquid cooling flow with the possibility of lowering the temperature of the cooled element to a value from 4 to 8 ° C. 12. The device according to claim 2, characterized in that the material of the decapitation cell is selected from glass or polymer. 13. The device according to p. 12, characterized in that the polymers are selected from the group consisting of polycarbonate, polymethyl methacrylate. 14. The device according to item 13, wherein the polymer is polymethylmethacrylate. 15. The device according to p. 13, characterized in that the material of the decapitation cell is made of a transparent material.