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WO2018124929A1 - Procédé de prélèvement de microparticules et de microtraces d'origine végétale et animale - Google Patents

Procédé de prélèvement de microparticules et de microtraces d'origine végétale et animale Download PDF

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Publication number
WO2018124929A1
WO2018124929A1 PCT/RU2017/000391 RU2017000391W WO2018124929A1 WO 2018124929 A1 WO2018124929 A1 WO 2018124929A1 RU 2017000391 W RU2017000391 W RU 2017000391W WO 2018124929 A1 WO2018124929 A1 WO 2018124929A1
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WIPO (PCT)
Prior art keywords
micro
microparticles
traces
plant
animal origin
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PCT/RU2017/000391
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English (en)
Russian (ru)
Inventor
Галина Евгеньевна ЛАРИНА
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

Definitions

  • 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 expertise.
  • a method for detecting the presence of submicron particles in a sample taken from the environment.
  • the method includes sampling from the environment, purification and concentration of 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.
  • this method is designed to capture particles smaller than 1 micron in size from air, which is much smaller than the size of organic dust particles, including pollen grains, whose size range is 10-100 microns.
  • 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 - 0 " 17 C.
  • This method is also not applicable for the collection of microparticles from deep pores, cracks in the surface of a macro object.
  • 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; exemption microparticles using ultrasonic cavitation in the environment of Freon-1 13.
  • the methods are used in medicine, forensic science, merchandising, palynomorphological and other studies (Gladkova AN, Grichuk V.P., Zaklinskaya E.D. et al. Pollen test, 1950 ; GOST 31769-2012 Medical method for determining the frequency of occurrence of pollen grains; Karevskaya I.A.
  • a method of sampling microparticles, i.e. pollen grains and spores from liquid substances, for example, honey 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 technical problem to which the claimed invention is directed is the insufficient quality of samples containing macro- and micro-objects, i.e. purity of samples in which there are many impurities of other microparticles, in addition to 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 5 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 results identification 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 surface active
  • the method can carry out additional washing of the material from deep 20 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 alcohol and alkali, followed by washing, drying and 25 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.
  • 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. Then, mild alkaline hydrolysis (a mixture of ammonia and alkali) is carried out during boiling, followed by washing, drying and staining of microparticles and micro-traces.
  • the proposed method is affordable, ergonomic and environmentally friendly.
  • FIG. 1-2 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 involves removing microparticles and micro-traces from the surface of the carrier object by the physicomechanical method on the orbital rocking chair (shaker) at a temperature of 20-25 ° C with a solution of monobasic carboxylic (acetic) acid with the addition of a surfactant, e.g. 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.
  • a surfactant e.g. Polysorbate 20
  • the second stage involves the removal of microparticles and micro-traces from the surface of the carrier object physically-mechanically in an ultrasonic bath at a temperature of 35-45 ° C with a solution of monobasic carboxylic (acetic) acid with the addition of Polysorbate 20 (liquid soap, TWEEN_20) .
  • sedimentation is carried out.
  • Alkaline hydrolysis of precipitates containing non-destroyed microobjects is carried out in an inert vessel in a boiling water bath with equal amounts of ammonia (NhUOH) and alkali (NaOH) added, followed by washing and drying of the precipitate by centrifugation.
  • NhUOH ammonia
  • NaOH alkali
  • the resulting precipitates with microobjects are stained with an alcohol solution of fuchsin and the preparation is fixed in a glycerin-containing medium for the microscopy procedure using light microscopy.
  • a precipitate is a component of a solution containing microparticles and micro-traces from a carrier object of biological origin.
  • the carrier object is placed in a container with a tight lid of inert material. All dishes are also used from inert material.
  • Example 1 A cardboard box from a pharmacy - Alder cones (fruits), 50g.
  • 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 was added to NsN ° 1-3 samples in containers with alder cones and 0.5 ml of Polysorbate 20 (TWEEN_20, liquid soap) was added. 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.
  • TWEEN_20 Polysorbate 20
  • Fat-free glass slide was placed on a heating table and
  • micromycetes 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-fold.
  • the palinoindication of pollen grains and spores of higher plants was carried out according to qualitative zoological attributes in accordance with descriptions in atlases, scientific literature, specialized interactive databases with descriptions and illustrations of microobjects. Three replicates were taken into account (NsNsl-3 sample), two glasses from each sample and at least 150 pollen grains (total number).
  • a + flush is (not) identifiable
  • Example 2 Plum seasonal, ordinary (sample weight 1 kg).
  • 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.
  • TWEEN_20 liquid soap
  • monobasic carboxylic (acetic) acid 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 centrifugal
  • L5 tubes (hereinafter - tubes) without plum fruit.
  • the saline tubes were centrifuged, i.e. carried out sedimentation for 25 min at a rotor speed of 3000 rpm./min in a laboratory centrifuge. After centrifugation, the tubes were carefully removed and the supernatant was drained.
  • 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.
  • the arithmetic mean value of the results of parallel determinations (sample N ° N ° 1-3) was taken as the final test result.
  • sample N ° N ° 1-3 the same sample (Plum, seasonal, ordinary) obtained by the same method, in the same laboratory, by the same laboratory assistant, using the same the same measuring instruments and equipment received the maximum permissible relative discrepancy of less than 15% of the arithmetic mean value (table 3).
  • 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%.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention se rapporte à l'étude et à l'analyse des matériaux et plus particulièrement à la production d'échantillons pour la recherche notamment de microparticules et de microtraces depuis un objet-substrat d'origine végétale et animale, et peut être utilisée en palynologie, en biologie, en écologie et en médecine pour l'analyse de spores et de pollens, ainsi que dans des expertises criminologiques, de marchandises et écologiques. Ce procédé de prélèvement de microparticules et de microtraces d'un objet d'origine végétale et animale depuis la surface d'un objet-substrat consiste à laver le matériau selon un procédé physique-mécanique à une température de 20-25°C dans une solution d'acide carboxylique à base unique en ajoutant une substance tensioactive pour effectuer une sédimentation. On effectue ensuite une hydrolyse alcaline du dépôt en utilisant un mélange d'alcool d'ammoniac et d'alcali, puis un rinçage, un séchage et une coloration des microparticules et des microtraces. Le résultat technique de l'invention consiste en une meilleure qualité des échantillons lors des étapes de préparation et en la production d'un ensemble complet maximal de microparticules et de microtraces grâce au choix des solvants et des modes de lavage, ce qui permet d'accélérer les travaux routiniers lors du prélèvement de microparticules et de microtraces d'origines végétale et animale d'un objet-substrat, et d'améliorer l'efficacité des résultats d'analyse d'identification dans l'ensemble.
PCT/RU2017/000391 2016-12-30 2017-06-08 Procédé de prélèvement de microparticules et de microtraces d'origine végétale et animale Ceased WO2018124929A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2016152567A RU2651171C1 (ru) 2016-12-30 2016-12-30 Способ сбора микрочастиц и микроследов с объекта растительного и животного происхождения
RU2016152567 2016-12-30

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112284861A (zh) * 2020-10-21 2021-01-29 上海市农业科学院 用于真姬菇担孢子显微观察的固定液、制备方法、固定方法及应用

Citations (4)

* Cited by examiner, † Cited by third party
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US20030199100A1 (en) * 2000-09-15 2003-10-23 Wick Charles Harold Method and system for detecting and recording submicron sized particles
RU2229109C2 (ru) * 2002-05-06 2004-05-20 Военно-морской инженерный институт Способ отбора и обработки проб для определения загрязненности поверхностей металлической ртутью и ее соединениями
RU2239837C2 (ru) * 2002-08-15 2004-11-10 Государственное учреждение системы высшего и послевузовского профессионального образования "Сибирский государственный медицинский университет" Способ оценки степени тяжести атопического дерматита
RU2402781C1 (ru) * 2009-06-23 2010-10-27 Государственное научное учреждение "Прикаспийский зональный НИВИ" Россельхозакадемии Способ предпосевной обработки проб, снятых с объектов внешней среды, на выделение микобактерий

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SU1735737A1 (ru) * 1989-08-31 1992-05-23 Научно-Исследовательский Институт Общей И Коммунальной Гигиены Им.А.Н.Сысина Способ подготовки эпителиальных клеток желудочно-кишечного тракта к исследованию
RU2370540C2 (ru) * 2007-08-06 2009-10-20 Автономная некоммерческая организация "Центр инноваций и наукоемких технологий" Способ определения биотоксичности питьевой минеральной воды

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030199100A1 (en) * 2000-09-15 2003-10-23 Wick Charles Harold Method and system for detecting and recording submicron sized particles
RU2229109C2 (ru) * 2002-05-06 2004-05-20 Военно-морской инженерный институт Способ отбора и обработки проб для определения загрязненности поверхностей металлической ртутью и ее соединениями
RU2239837C2 (ru) * 2002-08-15 2004-11-10 Государственное учреждение системы высшего и послевузовского профессионального образования "Сибирский государственный медицинский университет" Способ оценки степени тяжести атопического дерматита
RU2402781C1 (ru) * 2009-06-23 2010-10-27 Государственное научное учреждение "Прикаспийский зональный НИВИ" Россельхозакадемии Способ предпосевной обработки проб, снятых с объектов внешней среды, на выделение микобактерий

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112284861A (zh) * 2020-10-21 2021-01-29 上海市农业科学院 用于真姬菇担孢子显微观察的固定液、制备方法、固定方法及应用
CN112284861B (zh) * 2020-10-21 2023-02-17 上海市农业科学院 用于真姬菇担孢子显微观察的固定液、制备方法、固定方法及应用

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