SU1010128A1 - Method for indicating contamination of objects of environment with chemical substances - Google Patents

Abstract

1. METHOD OF INDICATION OF POLLUTION OF OBJECTS OF THE ENVIRONMENT BY CHEMICAL SUBSTANCES by affecting the sample on the gest culture of microorganisms with subsequent control of changes in the culture, characterized in that, in order to increase the sensitivity and shorten the display time, the sample is introduced into one end of the culture by passing the sample to the one end, if the sensitivity is increased and the display time is reduced, the sample is applied to the culture of microorganisms. a suspension of microorganisms exhibiting the effect of chemotaxis on the substances to be displayed, measures the redistribution of the concentration of microorganisms along the cuvette and the difference in the concentration of microorganisms in Contaminated areas determine the degree of contamination. (/) C - / 3 - / - / 7 -l -ff

Description

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2, the method according to claim 1, characterized in that the redistribution of the concentration of microorganisms in the cuvette is measured by the photometric method.

3. Method according to paragraphs. 1 and 2, characterized in that in the indication of contamination by heavy metals, microorganisms are used as microorganisms.

The invention relates to the environment, namely to the methods of the op. determine the degree of contamination of water, boadukh, and soil in the field of industrial and sanitary hygiene, and can be used for toxicological control in the food, chemical, pharmaceutical, and other industries.

Substances that pollute air, soil, water, include ammonia, sulfurous water. genus, salts of heavy metals (Cu, N.i H) and others.

Various methods are known for the determination of environmental pollution, including TOK-i using the simplest microorganisms.

There is a method of assessing the toxicity of waste and natural waters, including the incubation CfiEoreK a rugepozyoz 82 in the sample and the determination of the difference in growth in the experimental and control samples l.

The most suggested method is the determination of contamination with chemicals, judged by their toxicity level, which consists in introducing a sample of the test material into interaction with the test and inoculating it under conditions that support their good growth (experience). Then, the grown MHKpoopraHH3Na i is filtered from the liquid and their number is determined turbidimetrically. It is compared with the number of microorganisms grown in the counterpart without the test being examined, and the degree of toxicity of the material under study 3 is determined by the difference in growth in experiment and control.

The disadvantages of this method YAS are low sensitivity and duration of the analysis. The sensitivity of the Mtuia method, since the physiological processes of the xopoiuo cell are protected from nrblagopri ttlh impact, according to 8c (oiB8us mec oiterium to a concentration of os. / Ml.

4, The method according to PP "1 and 2, I differ from P1 and the fact that when indicating the loading of objects surrounded by gaseous sulfur compounds as microorganisms, RagsgleCiurn cau oi-tom; B KoHneHTpalimi from 1O to 1O units / ml are used as microorganisms.

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This requires high concentrations of toxicants to alter the growth rate of microorganisms.

The analysis time for the growth rate is determined by no less than twice the generation time of the test culture and reaches several hours and even days.

The aim of the invention is to increase the sensitivity and shorten the time of the pollution indication of environmental objects.

The goal is achieved by the fact that, according to the method of indicating contamination by chemical substances of environmental objects, which involves exposing the test culture of microorganisms to subsequent changes in the culture, the sample is introduced into one end of the extended cuvette filled with a suspension of microorganisms effect of chemotaxis on the displayed substances, measure the redistribution of the concentration of microorganisms along the cuvette and the difference in the concentration of microorganisms in controlled areas -determine dissolved degree of contamination.

The redistribution of the concentration of microorganisms in the cuvette is usually changed by the photometric method. In the indication of contamination with heavy metals, BaeiC (; 5 tnec qtBriUm at a concentration of 10-10 units / ml) are commonly used as microorganisms.

When indicating the contamination of environmental objects with gaseous sulfur compounds, use PotranieciuTri C "i 61a-beta concentration 1 GB units / ml:

The proposed method makes it possible to implement a fundamentally more sensitive and faster test reaction of microorganisms, chemotaxis, directed by dispersion A (1

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under the influence of chemical substances. Sensitivity and speed of this test reaction and the proposed methods, confirmation of the fact that this position interacts, the cells with chemicals occur on the re-table.

peptone level on the cell surface. 5 different test-response myeofoorganisms to

Comparative experimental data of chemicals.

the concentration required to kill 50% of the cells in J2 hours; the concentration of the substance required for 50% inhibition of cell growth for 1 and 4 days; the minimum concentration of the substance causing chemotaxis. The chemotactic reaction takes place only when there is a difference in the concentration of chemical substances along the cuvette, otherwise the movement of microorganisms is chaotic, and they are distributed evenly throughout the cuvette. The use of an elongated cuvette in the proposed method, the insertion of the test sample only at one end creates a difference in the maximization of the sample along the length of the cuvette, which causes the microorganisms to actively mix up at the end of the cuvette where the conditions for livelihood are more favorable. The redistribution of microorganisms in the cuvette can be measured by photometric, conductometric, or other methods. The distance at which it is possible to detect the redistribution of the concentrations of microorganisms is determined by the technical characteristics of the respective measuring devices and the size of the microorganisms and is about 2 mm. Bacteria can pass this distance in 18O with 3J, Pcirame ciumicaucldtumaa 0.8c 4 by counting the dif-fuan substances from the sample will penetrate into the suspension of microorganisms at a distance of 2 mm per 10 sec (calculated from the level of unsteady diffusion from a limited volume by the fastest ions). Therefore, microorganisms in all known cases can show the sample introduction zone faster than diffusion occurs. Thus, it takes only from 0.8 to 180 seconds to obtain in a cuvette from a site with a sufficiently pronounced difference in the concentration of microorganisms to measure. This time is determined by the rate of indication of the proposed method, /,: When implementing the method, all microorganisms with a chemotaxis effect on the displayed substances can be used. In each case, you can choose the microorganism that cat (it gives the greatest positive effect of the method. The large variety of microorganisms and their selectivity of the reaction to certain substances makes it possible to say that when solving the problem of indicating a certain substance, you can choose the appropriate culture. Poircimpcium txaudalutn was tested by Hfl cultures of Batieeus SuttiBiS, BociSBus mecjterium, Serraties rnarsences, Proteus. For all of the studied cultures, the possibility of indicating chemical contamination by the proposed method The method is carried out as follows: Prepare for analysis a sample in a liquid that is indifferent to the microorganisms used and does not cause chemotaxis. The water sample is diluted, if necessary, to a concentration that does not cause the test culture to die, it is diluted; the analyzed air is bubbled through the device. A standard suspension of microorganisms is prepared in an environment that maintains cell mobility during the indication time, for example, water, saline, and Motile microorganisms possessing hematoxis effect to indexable substances (algae, protozoa bacteria). Fill with a standard suspension of test culture. ud linear cuvette}, at one end of the test sample in the amount of 1/201/5 of the volume of suspension is introduced. Then, the change in the concentration of microorganisms in several sections of the cuvette is controlled by photometric, conductometric or apyiTiMi methods from the moment the sample is introduced to the end of the display, which is determined by the intensity of the chemotactic process, the nature of the chemicals and the speed of movement of the indicator microorganisms. Measurements are carried out continuously or discretely and the degree of contamination is estimated from the difference in the concentration of microorganisms along the length of the cell. There is no need to measure the absolute value of the concentration. It is sufficient to use indirect parameters characterizing the magnitude of the concentration of microorganisms, for example, optical density, electrical conductivity, etc. In FIG. Figure 1 shows the dependence of the magnitude of the chemotactic reaction (& E} on the concentration of ions (mol / l); in 4ng. 2 - dependence, magnitude of the chemotactic reaction (uJ) on the concentration of ions with (C mop / l); Fig. 3 the dependence of the chemotactic value of 5) action (LJ) on time (-t) with doyc VII ions and t {n concentration 2 -.I. About mol / l; Cha fig, -1 - S v1K ikok: t1. vipgsh8 of the chemotactic reaction (D3) against time (t) under the action of cG ions in a concentration of 1 mop / l; in fig. 5 dependence of the chemotactic reaction (/ LE), and on time at different concentrations of NH, and (5) on the concentration of NHj for 30 min of exposure; in fig. 6 - dependence of the chemotactic reaction (.DZ) on the time of the chemical effect of {a –Ni | 6-Cu) oT concentration of the chemical substance (6-Ni i-Cju) per D30 min of exposure. Example 1. It is carried out, the indionation of Hc ions, 5 the corresponding anion and the cation of which do not cause chemotaxis in the test culture, which was previously checked experimentally. The test concentrations of the tested ions are prepared in a medium. Lozina-Lozinskiy following composition, g / l: I-IO 1-10-4 KCY 1-10M SO4, -4 CaceaIlo NaHCO 32-10 The test culture of ParofmeciUfn ccnodatom is grown by the method 5 , removed from yeast by Lozin-Lozinsky medium in a centrifuge (3000 rpm, 5 min), added to a glass cell of size 3010-5 mm in an amount of 3 ml, a suspension of microorganisms with a concentration of ZOOO cells in 1 ml is used in grill. In the same cuvette, a displayed substance in the amount of 0.3 ml is introduced into one of its ends. The redistribution of the concentration of pqramecium CCHUdatvJm along the cuvette under the influence of the sample by the photometric method in two sections of the cuvette is measured, for which two photodetectors are used, placed: the first - 26 mm from the place of the sample introduction, the second - 52 mm. Measurements are carried out from the moment the sample is introduced for 30 minutes at intervals of 5 minutes. The degree of contamination is estimated by the difference of signals (flj) from the first (J) and second (ZU) photodetectors, since the magnitude of the signal is proportional to the concentration of microorganisms in the monitored areas. The sign of the magnitude of LE is characterized by a positive or negative direction of the chemotactic reaction, the F-Results of Hoj 5 ion indication are shown in Figs 1–4, The sensitivity threshold of the method: for the Htt ion 2 1 O 2 m / l (Fig); 10 ° mol / l (fi 2). for ion 3 The magnitude of the chemotactic reaction n; it is proportional to the concentration of chemically chemical substances in a wide range of the latter, namely: for 2 1 (2 mol / l; (Fig. 1) T for 5 from 1-1О to 1 -10 mol / L, (Fig. 2). The LE value reaches the value under the measurement by the proposed method after 2-5 minutes and increases during the entire display time (30 minutes in this example) (Fig. 3 and 4). For Htj, the magnitude of LE is negative, the microorganisms move away from the point of entry of the sample — negative chemotaxis (Figs. 1 and 3). LL ion3 C 3 — the magnitude of a3 is positive, microorganisms are about are collected, collected at the sample entry site, i.e., there is a positive chemotaxis (Figures 2 and 4). Example 2. Indication of air sample contamination. A common air contamination is ammonia, therefore air pollution with this substance. Prepared; mixture of air with ammonia. The mixture is bubbled through distilled water for 15 minutes. The concentration of ammonia in water is determined according to standard methdac (Nessler's method). Working solutions with a known concentration of ammonia are prepared in Lozin-Lozinsky medium by dilution to the concentration studied. Further, according to the method, testing is carried out according to the procedure given in Example 1. Ammonia solutions are examined in the concentration range 1.7 x 1.7 x 10 m / l. A higher concentration (1.7-K ol / l) caused the death of the Pctramecium sai culture. Rectum is therefore not determined by the proposed method. Samples with an ammonia concentration of 1.7-1 O 1 p 1 cause a negative chemotactic reaction, and with an ammonia concentration of 1, 7–10 a positive v chemotactic reaction, the magnitudes of which are determined by the proposed method from the difference in the concentration of microorganisms along the cell Density of the experiments is presented in the graphs of Fig. 5. EXAMPLE 3 Determination of Ni and SU ions in soil. The soil is modeled with washed sand. The sand mixes with weights of nickel and copper salts. The hinges of salts are based on their solubility in distilled Water. A mixture of sand and salt is placed in distilled water of a volatile volume and mixed. The salt solution is drained and working solutions of salts of a known concentration in Lozina-Lozinsky are prepared. Next, the test is carried out as described in Example 1. Display results are presented in the graphs of Fig. 6. Thus, tests have shown that the proposed method is highly sensitive (several orders of magnitude higher than the prototype) and shortens the display time from several hours to minutes. Similar results on the sensitivity and speed of the display by the proposed method are obtained on other displayed objects from water, air, soil. In addition, the proposed method makes it possible to expand the range of indicated substances through the use of a set of test cultures, as well as through the use of both positive and negative chemotaxis.

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Claims (4)

1. METHOD FOR INDICATING ENVIRONMENTAL POLLUTION BY CHEMICAL SUBSTANCES by affecting the sample on the test culture of microorganisms with subsequent control of changes in the culture, characterized in that, in order to increase sensitivity and reduce the time of indication, the sample is introduced at one end of an extended cell containing microorganism suspension showing the effect of chemotaxis to the indicated substances, measure the redistribution of the concentration of microorganisms in the distance and the difference in the concentration of microorganisms in the control iruemyh sites determine the extent of contamination. 2. The method according to π. 1, characterized in that the redistribution of the concentration of microorganisms in the cell is measured by the photometric method. 3. The method according to PP. 1 and 2, with πη- η and the fact that when indicating pollution by heavy metals as microorganisms use BaciKSus rneqjateriurn to a concentration of 10θ to 10® units / ml. 4. The method according to PP. 1 and 2, they are distinguished by the fact that when indicating the pollution of environmental objects with gaseous sulfur compounds, ParcftneCiurn cctueJortu'nv in concentration of 10 * to 10 * units, / ml is used as microorganisms.