RU2651171C1 - Method of collecting micro-particles and micro-traces from the objects of vegetable and animal origin - Google Patents

Abstract

FIELD: analysis of materials.

SUBSTANCE: invention relates to the examination or analysis of materials, namely, to the preparation of samples for examination, in particular, microparticles and micro traces from a carrier object of plant and animal origin, and can be used in palynology, biology, ecology, medicine for spore-pollen analysis, forensic, commodity and ecological expert reviews. From the carrier object surface, the material is washed away physically and mechanically at a temperature of 20–25 °C in a monobasic carboxylic acid solution with the addition of a surfactant followed by sedimentation. Then alkaline hydrolysis of the precipitate is carried out using an ammonia and alkali mixture, followed by washing, drying and staining of microparticles and micro-traces.

EFFECT: enabling improvement of the samples quality at the preparatory stages and obtaining the most complete set of microparticles and micro-traces due to the selection of solvents and flushing regimes, which makes it possible to speed up routine work when removing microparticles and micro-traces of plant and animal origin from the carrier object, improve the effectiveness of the identification analysis results as a whole.

1 cl, 2 dwg, 2 ex, 3 tbl

Description

The invention relates to the study or analysis of materials, namely, to obtain samples for research, in particular microparticles and micro-traces from a carrier object of plant and animal origin, and can be used in palynology, biology, ecology, medicine for spore-pollen analysis, forensic, merchandising and environmental reviews.

From US Pat. No. 7,250,138, a method is known for detecting the presence of submicron particles in a sample taken from the environment. The method includes sampling from the environment, cleaning and concentrating submicron particles in the sample based on particle size. The purified and concentrated particles are detected using a device that includes an electrospray assembly having a capillary, a differential mobility analyzer that receives an output signal from the capillary, and a particle condensation device for counting the number of particles that pass through the differential mobility analyzer.

However, this method is designed to capture particles smaller than 1 μm in size from air, which is much smaller than the size of organic dust particles, including pollen grains, whose size range is 10-100 μm. The method employs a device - an electrospray assembly. The pollen of higher plants is characterized by the presence of an electric charge, but one flower contains a different number of pollen grains charged both positively and negatively. The charge of pollen is extremely small and is in the range of 10 ^-16 -10 ^-17 C. This method is also not applicable for the collection of microparticles from deep pores, cracks in the surface of a macro object.

There is also known a method of grinding, extraction and detection of analytes in solid biological samples from international application WO 2005063962. This invention relates to methods, reagents, kits, devices and automated systems for grinding, extraction and detection of drug analytes, pesticides, herbicides and steroids in solid biological samples originating, for example, from humans, domestic animals, plants, amphibians, insects and reptiles.

However, this method is characterized by high economic costs for electricity, water and the disposal of toxic waste, where, for example, hydrochloric acid is used as a solvent. In the extraction and detection of analytes, automated chromatographic systems, expensive foreign KIT kits (KitLab) and analytical standards of high purity are used. In addition, the use of grinding at the stage of preparation of the preparations leads to the complete destruction of microobjects, including pollen grains and spores, without the possibility of their registration and identification by morphometric characteristics.

Also known is a technical solution from patent RU 2273853 "Method for bacterioscopic rapid identification of microflora of the contents of the colon and rectum." This invention relates to the field of medicine - coloproctology, gastroenterology, clinical microbiology. The method involves sampling, which is diluted with distilled water, the averaged suspension of the substrate is defended, 95.5 ° ethanol is added, centrifuged in two stages, a monolayer smear is made from the precipitate, which is dried at room temperature, fixed at 95.5 ° ethanol, then stained Gram smear and determine the morphology of microorganisms. The invention provides quick identification of a large number of species of microbial populations while simultaneously determining the ratios of gram-positive and gram-negative forms.

However, the average size of bacteria is 0.5-5 microns, which is much smaller than the size of pollen grains. Therefore, this method is not applicable for macroobjects of plant and animal origin, because it does not allow to separate large “garbage” - pollen grains and spores - from bacterial cells in full. In addition, pollen grains and spores in excrement should not be normal, because this indicates a violation of peristalsis and too rapid evacuation of food masses from the intestine.

The closest in technical essence to the proposed method is a method of removing microparticles and micro-traces from objects-carriers by the vacuum method (for example, a vacuum cleaner, special nozzles, filters); removal of microparticles by ultrasound in inert environments; the removal of microparticles using ultrasonic cavitation in the environment of Freon-113. The methods are used in medicine, forensic science, commodity science, palynomorphological and other studies (Gladkova AN, Grichuk VP, Zaklinskaya ED and others. Pollen analysis, 1950; GOST 31769-2012 Medical method for determining the frequency of occurrence of pollen grains; Karevskaya IA Spore-pollen analysis in paleogeographic and geomorphological studies, 1999; Dodonkin Yu.V., Zhebeleva IA, Krishtafovich VI Customs examination of goods, 2004; GOST 28887-90. Flower pollen ( shredding). Technical specifications; GOST R 51074-2003. Food products; etc.).

A method of sampling microparticles, i.e. pollen grains and spores from liquid substances, such as honey (GOST 28887-90, GOST 31769-2012), are based on the addition of distilled water to the sample and centrifugation in two stages. The precipitate is stained with a 0.1% alcohol solution of fuchsin. Then the drug is fixed in glycerol-gelatin and the morphology of pollen grains is determined. This method is operational and provides identification of a large number of species of pollen grains and spores from liquid media, but is not applicable for removing microobjects from the surface and roughnesses of a carrier object. The vacuum method and ultrasonic cavitation as methods of removing microobjects, along with widespread use and time testing, also have a number of significant limitations. Firstly, it is problematic for medical aspects (there are restrictions) - noise pollution and additional dusting, compliance with strict requirements for sanitary standards, including freon. Secondly, the economic costs of resources (electricity) and the time of routine work, as well as the disposal of toxic waste, which have a significant impact on the ecology of the workplace. Thirdly, this method does not provide a sufficient set (or range) of microparticles, because many of them are destroyed or lost during extraction, grinding and other procedures. Fourth, when implementing this method, it is difficult, and sometimes impossible, to remove microparticles from a carrier object of plant and animal origin for subsequent registration / identification.

The technical problem to which the claimed invention is directed is the insufficient quality of samples containing macro- and micro-objects, i.e. the purity of the samples, in which there are many impurities of other microparticles, except pollen grains and spores, the destruction of micro-objects to unidentifiable residues in the process of removal, separation and preparation (alkaline hydrolysis) for registration.

The technical result of the claimed invention is to improve the quality of samples at the preparatory stages and to obtain the most complete set of microparticles and microseeds due to the choice of solvents and flushing modes, which allows to accelerate routine work when removing microparticles and microseeds of plant and animal origin from the carrier object, to increase the efficiency of the identification results analysis in general.

The specified technical result is achieved due to the fact that in the method of removing microparticles and micro-traces from an object of plant and animal origin from the surface of the carrier object, the material is washed off physically-mechanically at a temperature of 20-25 ° C in a solution of monobasic carboxylic acid with the addition of surfactant substances with subsequent sedimentation, after which alkaline hydrolysis of the precipitate is carried out using a mixture of ammonia and alkali, followed by washing, drying and staining with microns particles and micro traces.

The method can carry out additional flushing of material from deep layers of the surface of the carrier object under ultrasonic treatment at a temperature of 35-45 ° C in a solution of monobasic carboxylic acid with the addition of a surfactant with further sedimentation, after which alkaline hydrolysis of the precipitate is carried out using a mixture of ammonia and alkali, followed by washing, drying and staining of microparticles and micro-traces.

The achievement of the indicated technical result is ensured by using the non-destructive principle of removing microparticles and micro-traces of plant and animal origin from the carrier object. In the method, the material is washed off at room temperature (20-25 ° C) in a solution of monobasic carboxylic acid with the addition of a surfactant and subsequent sedimentation. After that, a soft alkaline hydrolysis (a mixture of ammonia and alkali) is carried out during boiling, followed by washing, drying and staining of microparticles and micro trace. The proposed method is affordable, ergonomic and environmentally friendly.

The implementation of the invention is illustrated by the flowcharts in FIG. 1-2, which show:

FIG. 1 is an algorithm for implementing the method with the main flush;

FIG. 2 is an algorithm for implementing the method with an additional flush.

The method is implemented as follows.

The first stage (flushing A or the main one) involves removing microparticles and micro-traces from the surface of the carrier object physically-mechanically on an orbital shaker (shaker) at a temperature of 20-25 ° C with a solution of monobasic carboxylic (acetic) acid with the addition of a surfactant, for example Polysorbate 20 (liquid soap, TWEEN_20). Sedimentation of aqueous solutions is carried out with the addition of sodium chloride at a temperature of 20-25 ° C in a laboratory centrifuge.

The second stage (flushing B or additional) involves the removal of microparticles and micro-traces from the surface of the carrier object by the physicomechanical method in an ultrasonic bath at a temperature of 35-45 ° С with a solution of monobasic carboxylic (acetic) acid with the addition of Polysorbate 20 (liquid soap, TWEEN_20). Next, sedimentation of aqueous solutions is carried out in a laboratory centrifuge at room temperature.

Alkaline hydrolysis of precipitates containing non-destroyed micro-objects is carried out in an inert vessel in a boiling water bath with the addition of equal amounts of ammonia (NH ₄ OH) and alkali (NaOH), followed by washing and drying of the precipitate by centrifugation.

Next, the resulting precipitates with microobjects are stained with an alcoholic solution of fuchsin and the preparation is fixed in a glycerin-containing medium for the microscopy procedure using light microscopy.

Depending on the type of surface of the macroobject, an additional (independent) flushing of microobjects from deep layers of the surface of the carrier object is possible. Conditional division is accepted: type 1 - uneven, rough, pubescent, hard, rough, rough, wax / paraffin coating; type 2 - smooth, smooth, silky (table 1).

The following abbreviations are used in table 1: Ш - shaker, UZV - ultrasonic bath, BV - water bath, CF1 - centrifuge, CF2 - microcentrifuge-vortex, OH - carrier object.

It is understood that a precipitate is a component of a solution containing microparticles and micro-traces from a carrier object of biological origin.

Note:

To remove microparticles, the carrier object is placed in a container with a tight lid of inert material. All dishes are also used from inert material.

At the “Flush A” stage, 1 cm ^{3 of a} 20% solution of monobasic carboxylic (acetic) acid is added to one volume of the carrier object equal to 1 cm ³ , i.e. 50 ml of the solution is added to the carrier object with an average volume of 50 cm ³ , for example, root crops, berries, etc.

At the “Flush B” stage, 2 cm ^{3 of a} 10% solution of monobasic carboxylic (acetic) acid is added to one volume of the carrier object equal to 1 cm ³ .

Examples of the use of this method

Example 1. A cardboard box from a pharmacy - alder cones (fruits), 50 g.

Test preparation

Flushing A. A 50-g sample of alder fruit was taken from the package and placed in a container with a tight lid. Then, 0.5 ml of Polysorbate 20 (TWEEN_20, liquid soap) was added, 50 ml was poured with a 20% solution of monobasic carboxylic (acetic) acid and placed on an orbital shaker (shaker) at a temperature of 20-25 ° C for 30 min with a frequency of 1000 rpm One volume of a solution of a monobasic carboxylic (acetic) acid, i.e. 50 ml of the solution was added per 50 g. The solution was left to stand for 10 minutes and the supernatant was poured into centrifuge tubes (hereinafter - tubes) without alder fruit. 5 g of sodium chloride was added to the solution in the tubes and centrifuged, i.e. carried out sedimentation for 15 min at a rotor speed of 3000 rpm in a laboratory centrifuge. After centrifugation, the tubes were carefully removed and the supernatant was drained.

10 ml of the mixture was added to the precipitate in vitro: in equal volumes of 10% ammonia (NH ₄ OH) and 10% alkali (NaOH) and alkaline hydrolysis of the obtained products was carried out for 10 min in a water bath at 95-99 ° С sediments containing undestructed microobjects. Then the tubes were centrifuged, i.e. sedimentation was carried out for 10 min at a rotor speed of 5000 rpm in a laboratory centrifuge, the supernatant (alkali) was drained and washed with water, adding 10 ml of distilled water to the test tube, stirring with a glass rod, and centrifuged for 10 min at rotor speed of 5000 rpm. The procedure for washing the precipitate with water was repeated. Then the supernatant (supernatant) was drained and 1 ml of distilled water was added to the precipitate, mixed on a vortex microcentrifuge for 10 s and transferred to an 2 ml eppendorf tube. The suspension was centrifuged on a vortex microcentrifuge for 10 min at an acceleration of 1000 g. 0.03 ml of sediment suspension was taken carefully from the supernatant using an automated dispenser, transferred to an eppendorf tube of 0.5 ml volume, 2-3 drops of a 0.1% fuchsin alcohol solution were added, i.e. 10 mg of basic fuchsin per 60 ml of 96% alcohol, for staining and shaken on a vortex.

A fat-free glass slide was placed on a heating table and a circle of wax was applied. One drop of the suspension with microobjects was placed in the center and covered with a coverslip. The finished product was microscopic using a professional biological light microscope.

Flushing B. After flushing A was obtained, 100 ml of a 10% solution of monobasic carboxylic (acetic) acid were added to containers with cones of alder 100 # 1-3, and 0.5 ml of Polysorbate 20 (TWEEN_20, liquid soap) was added to containers with alder cones. Two volumes of a solution of a monobasic carboxylic (acetic) acid, i.e. per 50 g was added 100 ml of solution. The containers were tightly closed and placed on an ultrasonic bath for 10 min with maximum power, then they settled for 10 min and the supernatant was poured into centrifuge tubes (hereinafter - tubes) without alder fruit. To the solution, 5 g of sodium chloride was added to the tubes and centrifuged by sedimentation for 15 min at a rotor speed of 3000 rpm in a laboratory centrifuge. After centrifugation, the tubes were carefully removed and the supernatant was drained.

10 ml of the mixture was added to the precipitate in vitro — in equal volumes of 10% ammonia (NH ₄ OH) and 10% alkali (NaOH), and alkaline hydrolysis of the obtained products was carried out for 10 min in a water bath at 95-99 ° С sediments containing undestructed microobjects. Then the tubes were centrifuged by sedimentation for 10 min at a rotor speed of 5000 rpm in a laboratory centrifuge. The supernatant (alkali) was drained and washed with water, adding 10 ml of distilled water to the test tube, stirring with a glass rod, and centrifuged for 10 min at a rotor speed of 5000 rpm. The procedure for washing the precipitate with water was repeated. Then the supernatant (supernatant) was drained and 1 ml of distilled water was added to the precipitate, mixed on a vortex microcentrifuge for 10 s and transferred to an 2 ml eppendorf tube. The suspension was centrifuged on a vortex microcentrifuge for 10 min at an acceleration of 1000 g. 0.03 ml of sediment suspension was taken from the supernatant using an automated dispenser, 0.5 ml of eppendorf was transferred to a test tube, 2-3 drops of a 0.1% alcohol solution of fuchsin were added: 10 mg of basic fuchsin per 60 ml of 96% alcohol for staining and shaken on a microcentrifuge-vortex.

A fat-free glass slide was placed on a heating table and a circle of wax was applied. One drop of the suspension with microobjects was placed in the center and covered with a coverslip. The finished product was microscopic using a professional biological light microscope.

Test

Registration and analysis of microobjects - the total number of pollen grains and the number of pollen grains of certain genera / species, micromycetes - was carried out using light microscopy with an increase of 40-1000 times. The palinoindication of pollen grains and spores of higher plants (micromycetes, phytoliths, etc. may be indicated) was carried out according to qualitative characteristics in accordance with descriptions in atlases, scientific literature, specialized interactive databases with descriptions and illustrations of microobjects. Three replicates were taken into account (sample No. 1-3), two glasses from each sample and at least 150 pollen grains (total number).

Test Results Processing

        The number of pollen grains of the determined genus / species of the plant X,%, was calculated by the formula: X = a / b * 100%, where a is the number of pollen grains of the determined genus / species in the preparation, pcs .; b is the total number of pollen grains recorded in the preparation, pcs .; 100 - conversion factor to the mass fraction (%) of pollen grains of a defined genus / species. For the final test result, the arithmetic mean of the results of parallel determinations was taken (hinge No. 1-3).

As a result, between the three test results (sample No. 1-3) of one and the same sample (alder cones (fruits), 50 g, a cardboard box from a pharmacy), obtained according to the same method, in the same laboratory, by the same the laboratory assistant, using the same measuring instrument and equipment, received the maximum permissible relative discrepancy of less than 20% of the arithmetic mean value (table 2).

Example 2. Plum seasonal, ordinary (sample weight 1 kg).

Test preparation

Flushing A. We took three samples in each of three plum fruits and placed in a container with a tight lid. The weight of the plum depends on the variety and size of the crop, but the average weight is 30 g, i.e. each sample is 90-100 g. 0.5 ml of Polysorbate 20 (TWEEN_20, liquid soap) and 100 ml of a 20% solution of monobasic carboxylic (acetic) acid are added to each sample. For better removal of microobjects by mechanical friction and separation from the carrier object, 5 g of sodium chloride was added. One volume of a solution of a monobasic carboxylic (acetic) acid, i.e. per 100 g was added 100 ml of solution. A container with a tightly closed lid was placed on an orbital rocking chair (shaker) at a temperature of 20-25 ° C for 30 min with a frequency of 1000 rpm. The solution was left to stand for 10 min and the supernatant was poured into centrifuge tubes (hereinafter - tubes) without plum fruits.

The saline tubes were centrifuged, i.e. carried out sedimentation for 25 min at a rotor speed of 3000 rpm in a laboratory centrifuge. After centrifugation, the tubes were carefully removed and the supernatant was drained.

10 ml of the mixture was added to the precipitate in vitro — in equal volumes of 10% ammonia (NH ₄ OH) and 10% alkali (NaOH) —and alkaline hydrolysis was carried out for 10 min in a water bath at 95-99 ° С received sediments containing undestructed microobjects. Then the tubes were centrifuged by sedimentation for 10 min at a rotor speed of 5000 rpm in a laboratory centrifuge, the supernatant (alkali) was drained and washed with water, adding 10 ml of distilled water to the tube, stirring with a glass rod, and centrifuged in for 10 minutes at a rotor speed of 5000 rpm. The procedure for washing the precipitate with water was repeated. Then the supernatant (supernatant) was drained and 1 ml of distilled water was added to the precipitate, mixed on a vortex microcentrifuge for 10 s and transferred to an 2 ml eppendorf tube. The suspension was centrifuged on a vortex microcentrifuge for 10 min at an acceleration of 1000 g. 0.03 ml of sediment suspension was taken carefully from the supernatant using an automated dispenser, transferred to an eppendorf tube of 0.5 ml volume, 2-3 drops of a 0.1% fuchsin alcohol solution were added, i.e. 10 mg of basic fuchsin per 60 ml of 96% alcohol for staining, and shaken on a micro-centrifuge-vortex.

A fat-free glass slide was placed on a heating table and a circle of wax was applied. In the center was placed one drop of a suspension with microobjects, covered with a coverslip. The finished product was microscopic using a professional biological light microscope.

Test

Registration and analysis of microobjects — the total number of pollen grains and the number of pollen grains of individual genera / species — was carried out using light microscopy with an increase of 40-1000 times. The palinoindication of pollen grains and spores of higher plants (micromycetes, phytoliths, etc. may be indicated) was carried out according to qualitative characteristics in accordance with descriptions in atlases, scientific literature, specialized interactive databases with descriptions and illustrations of microobjects. Three replicates are taken into account (sample No. 1-3), two glasses from each sample, at least 150 pollen grains (total number).

Test Results Processing

The number of pollen grains of the determined genus / species of the plant X,%, was calculated by the formula: X = a / b * 100%, where a is the number of pollen grains of the determined genus / species in the preparation, pcs .; b is the total number of pollen grains recorded in the preparation, pcs .; 100 - conversion factor to the mass fraction (%) of pollen grains of a defined genus / species. For the final test result, the arithmetic mean of the results of parallel determinations was taken (hinge No. 1-3).

As a result, between the three test results (sample No. 1-3) of the same sample (seasonal, ordinary plum) obtained by the same method, in the same laboratory, by the same laboratory assistant, using the same measuring instruments and equipment received the maximum permissible relative discrepancy of less than 15% of the arithmetic mean value (table 3).

It is obvious to a person skilled in the art that the claimed ranges of temperature and time are not alternatives, but represent a specific mode, which can vary within the specified limits depending on the source object, equipment used and other external factors.

The above examples are special cases and do not exhaust all possible implementations of the claimed invention.

The present invention is an affordable, ergonomic and environmentally friendly way to remove microparticles to obtain "clean" high-quality samples with a set of microparticles. The method allows to obtain the most complete set of microparticles and micro-traces of plant and animal origin, including pollen grains and spores of higher plants, palynomorphs, mycelium, bacteria, protozoa, phytoliths, fragments of plant and animal origin to characterize the properties of the carrier object. The method also allows to obtain a differentiated set of microparticles and micro-traces of plant and animal origin, taking into account the growth time of the carrier object, because deep or fixed microparticles settled in the place of growth of the carrier object are removed with an additional or hot wash; main or cold flush - later micro-traces associated with the packaging and movement of the carrier object. Using the proposed method speeds up routine work - the average time of work is 2.5 hours per 1 sample, and also increases the effectiveness of the results of identification analysis - the number of hard or unidentifiable micro-objects ranges from 1-8%.

Claims (2)

1. A method of collecting samples of microparticles and micro-traces from objects of plant and animal origin, characterized in that the material is washed off from the surface of the carrier by the physicomechanical method at a temperature of 20-25 ° C in a solution of monobasic carboxylic acid with the addition of a surfactant subsequent sedimentation, after which alkaline hydrolysis of the precipitate is carried out using a mixture of ammonia and alkali, followed by washing, drying and staining of microparticles and micro-traces. 2. The method according to p. 1, characterized in that they carry out additional flushing of the material from the deep layers of the surface of the carrier object under ultrasonic treatment at a temperature of 35-45 ° C in a solution of monobasic carboxylic acid with the addition of a surfactant.

Images